U.S. patent application number 11/910943 was filed with the patent office on 2012-01-26 for activation of sodium potassium atpase.
Invention is credited to Kai Yuan Xu.
Application Number | 20120020956 11/910943 |
Document ID | / |
Family ID | 37087533 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120020956 |
Kind Code |
A1 |
Xu; Kai Yuan |
January 26, 2012 |
ACTIVATION OF SODIUM POTASSIUM ATPASE
Abstract
Activation sites on the alpha subunit of sodium potassium ATPase
have been discovered. It has also been discovered that certain
antibodies that bind to the alpha subunit of sodium potassium
ATPase dramatically increase enzyme activity. There has never
before been a report of precise activation sites or drug
interaction sites for sodium potassium ATPase. Certain methods have
also been discovered for treating or preventing diseases associated
with low sodium potassium ATPase activity by administering
antibodies, antibody fragments and small molecules that bind to the
activation sites on the alpha subunit of sodium potassium
ATPase.
Inventors: |
Xu; Kai Yuan; (Cockeysville,
MD) |
Family ID: |
37087533 |
Appl. No.: |
11/910943 |
Filed: |
April 7, 2006 |
PCT Filed: |
April 7, 2006 |
PCT NO: |
PCT/US2006/012912 |
371 Date: |
September 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60669479 |
Apr 8, 2005 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1; 424/184.1; 424/185.1; 530/387.9 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
11/00 20180101; C12N 9/14 20130101; A61P 3/04 20180101; A61P 25/00
20180101; C07K 7/08 20130101; G01N 2333/914 20130101; A61P 21/00
20180101; A61P 9/00 20180101; A61P 27/00 20180101; C07K 7/50
20130101; A61P 13/00 20180101; C07K 7/64 20130101; A61P 3/00
20180101; A61P 37/04 20180101; A61P 43/00 20180101; C07K 7/04
20130101; A61P 3/10 20180101; A61K 2039/505 20130101; A61P 27/02
20180101; C07K 7/02 20130101; G01N 2800/325 20130101; G01N 2500/04
20130101; A61P 13/12 20180101; A61P 1/00 20180101; A61K 39/00
20130101; C07K 2317/75 20130101; A61P 25/28 20180101; A61P 9/04
20180101; A61K 39/0005 20130101; A61P 35/00 20180101; C07K 16/40
20130101; A61P 7/00 20180101; A61P 27/12 20180101 |
Class at
Publication: |
424/133.1 ;
530/387.9; 424/139.1; 424/185.1; 424/184.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61K 48/00 20060101
A61K048/00; A61P 3/10 20060101 A61P003/10; A61P 11/00 20060101
A61P011/00; A61P 13/00 20060101 A61P013/00; A61P 7/00 20060101
A61P007/00; A61P 1/00 20060101 A61P001/00; A61P 27/12 20060101
A61P027/12; A61P 9/12 20060101 A61P009/12; A61P 25/28 20060101
A61P025/28; A61P 27/02 20060101 A61P027/02; A61P 9/00 20060101
A61P009/00; A61P 43/00 20060101 A61P043/00; A61P 35/00 20060101
A61P035/00; A61P 13/12 20060101 A61P013/12; A61P 25/00 20060101
A61P025/00; A61P 3/04 20060101 A61P003/04; A61P 37/04 20060101
A61P037/04; A61P 21/00 20060101 A61P021/00; A61P 9/04 20060101
A61P009/04; C07K 16/40 20060101 C07K016/40 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] Parts of this invention were made with Government support
under Contract No. HL52175 awarded by NIH/NHLBI. The Government may
have certain rights in the invention.
Claims
1. A pharmaceutical composition that increases sodium-potassium
ATPase activity in an animal cell or bodily fluid, comprising an
antibody, antibody fragment or small molecule that binds to an
activation site on the alpha subunit of any isoform of sodium
potassium ATPase, which activation site includes DVEDSYGQQWTYEQR,
RSATEEEPPNDD, KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN,
and fragments, derivatives or variants thereof.
2. The pharmaceutical composition of claim 1, wherein the animal
cell is a member selected from the group comprising hepatocytes,
kidney cells, red blood cells, endothelial cells, lung cells, nerve
cells, lens cells, brain cells, muscle cells, cardiac myocytes, and
any cells that express sodium potassium ATPase.
3. A pharmaceutical composition for treating or preventing a
disease associated with low sodium-potassium ATPase expression or
activity in an animal cell or bodily fluid, comprising an antibody,
antibody fragment or small molecule that binds to an activation
site on the alpha subunit of any isoform of sodium potassium
ATPase, which activation site includes DVEDSYGQQWTYEQR,
RSATEEEPPNDD, KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN,
and fragments, derivatives or variants thereof.
4. The pharmaceutical composition of claim 3, wherein the animal
cell is a member selected from the group comprising hepatocytes,
kidney cells, red blood cells, endothelial cells, lung cells, nerve
cells, lens cells, brain cells, muscle cells, cardiac myocytes, and
any cells that express sodium potassium ATPase.
5. The pharmaceutical composition of claim 1, wherein the antibody
is a member selected from the group comprising, SSA78, SSA95,
SSA97, SSA401 and SSA412 and fragments thereof.
6. The pharmaceutical composition of claim 3, wherein the antibody
is a member selected from the group comprising, SSA78, SSA95,
SSA97, SSA401 and SSA412 and fragments thereof.
7. The pharmaceutical composition of claim 3, wherein the disease
is a member selected from the group comprising diabetes, lung
diseases, liver diseases, urinary tract diseases, hemorrhagic
shock, gastrointestinal diseases including colitis, cataracts,
hypertension, Alzheimer's disease, eye disease, heart disease,
aging, cancer, kidney diseases, obesity and diseases of the nervous
system.
8. An isolated antibody or antibody fragment that recognizes and
binds to an epitope comprising an amino acid sequence
HLLGIRETWDDRWIN or to fragments, derivatives or variants thereof in
the alpha subunit of any isoform of sodium potassium ATPase in an
animal.
9. The antibody or antibody fragment of claim 8, wherein binding of
the antibody to the alpha subunit of any isoform of sodium
potassium ATPase increases myocyte intracellular diastolic and
systolic calcium.
10. The antibody or antibody fragment of claim 8, wherein binding
of the antibody to the alpha subunit of any isoform of sodium
potassium ATPase exerts a positive inotropic effect in cardiac
myocytes.
11. The antibody or antibody fragment of claim 8, wherein the
antibody is used to treat or prevent heart disease and muscle
contractile disorders.
12. The antibody or antibody fragment of claim 8, wherein the
antibody is a monoclonal antibody.
13. The antibody or antibody fragment of claim 8, wherein the
antibody is a humanized antibody.
14. The antibody or antibody fragment of claim 8, wherein the
antibody is a polyclonal antibody.
15. The antibody or antibody fragment of claim 8, wherein the
antibody is a SSA401.
16. Methods for increasing the activity of any isoform of
sodium-potassium ATPase in an animal cell or bodily fluid,
comprising administering to the animal an antibody, an antibody
fragment or a small molecule that recognizes and binds to an
epitope in the alpha subunit of any isoform of sodium-potassium
ATPase, which epitope comprises an amino acid sequence that is a
member selected from the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives and variants thereof, and which antibody,
antibody fragment or small molecule is administered in an amount
that increases the activity of the sodium-potassium ATPase in the
cell or bodily fluid.
17. The method of claim 16, wherein the antibody is a member
selected from the group comprising, SSA78, SSA95, SSA97, SSA401 and
SSA412 and antibody fragments thereof.
15. The method as in claim 16, wherein the antibody is a polyclonal
antibody.
16. The method as in claim 16, wherein the antibody is a monoclonal
antibody.
17. The method as in claim 16, wherein the antibody is a humanized
antibody
18. The method as in claim 16, wherein the animal cell is a member
selected from the group comprising hepatocytes, kidney cells, red
blood cells, endothelial cells, lung cells, nerve cells, lens
cells, brain cells, muscle cells, cardiac myocytes, and any cells
that express sodium potassium ATPase.
19. The method of claim 16, wherein the antibody, antibody fragment
or small molecule is administered locally.
20. The method of claim 16, wherein the antibody, antibody fragment
or small molecule is administered systemically.
21. A method for treating or preventing a disease in an animal that
is associated with low expression or activity of any isoform of
sodium-potassium ATPase in a cell or bodily fluid, comprising
administering to the animal an antibody, antibody fragment or small
molecule that recognizes and binds to an epitope in the alpha
subunit of the sodium-potassium ATPase, which epitope comprises an
amino acid sequence that is a member selected from the group
comprising DVEDSYGQQWTYEQR, VPAISLAYEQAESD, KRQPRNPKTDKLVNE,
RSATEEEPPNDD, HLLGIRETWDDRWIN, and fragments, derivatives and
variants thereof, and which antibody, antibody fragment or small
molecule is administered in an amount that increases
sodium-potassium ATPase activity in the cell or bodily fluid
thereby preventing or treating or preventing the disease.
22. The method of claim 21, wherein the cell is a member selected
from the group comprising hepatocytes, kidney cells, red blood
cells, endothelial cells, lung cells, nerve cells, lens cells,
brain cells, muscle cells, cardiac myocytes, and any cells that
express sodium potassium ATPase.
23. The method of claim 21, wherein the disease is a member
selected from the group comprising diabetes, lung diseases, liver
diseases, urinary tract diseases, hemorrhagic shock,
gastrointestinal diseases including colitis, cataracts,
hypertension, Alzheimer's disease, eye disease, heart disease,
aging, cancer, kidney diseases, obesity and diseases of the nervous
system.
24. The method of claim 21, wherein the antibody, antibody fragment
or small molecule is administered systemically.
25. The method of claim 21, wherein the antibody, antibody fragment
or small molecule is administered locally.
26. The method of claim 21, wherein the antibody is a member
selected from the group comprising, SSA78, SSA95, SSA97, SSA401 and
SSA412.
27. A method for treating or preventing a disease in an animal that
is associated with low expression or activity of any isoform of
sodium-potassium ATPase in a cell or bodily fluid, comprising
administering to the animal one or more peptides selected from the
group comprising DVEDSYGQQWTYEQR, VPAISLAYEQAESD, KRQPRNPKTDKLVNE,
RSATEEEPPNDD, HLLGIRETWDDRWIN, and fragments, derivatives and
variants thereof, which peptides are administered in an amount that
stimulates the animal's immune system to produce antibodies that
recognize and bind to the respective peptide epitopes on the alpha
subunit of the sodium potassium ATPase thereby increasing ATPase
activity in the cell or bodily fluid to treat or prevent the
disease.
28. The method of claim 27, wherein the peptide is administered to
the animal systemically.
28. The method of claim 27, wherein the peptide is administered to
the animal locally.
29. The method of claim 27, wherein the disease is a member
selected from the group comprising diabetes, lung diseases, liver
diseases, urinary tract diseases, hemorrhagic shock,
gastrointestinal diseases including colitis, cataracts,
hypertension, Alzheimer's disease, eye disease, heart disease,
aging, cancer, kidney diseases, obesity and diseases of the nervous
system.
30. The method of claim 27, wherein the cell is a member selected
from the group comprising hepatocytes, kidney cells, red blood
cells, endothelial cells, lung cells, nerve cells, lens cells,
brain cells, muscle cells, cardiac myocytes, and any cells that
express sodium potassium ATPase.
31. A method for increasing the activity of any isoform of sodium
potassium ATPase in an animal cell or bodily fluid, comprising
administering a vector that carries a gene encoding one or more
peptides selected from the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives and variants thereof, which peptides are
expressed in an amount that stimulates the animal's immune system
to produce antibodies that recognize and bind to the respective
peptide epitopes on the alpha subunit of the sodium potassium
ATPase thereby increasing the activity of the sodium potassium
ATPase in the cell or bodily fluid.
32. A method for preventing or treating or preventing a disease in
an animal that is associated with low sodium-potassium ATPase
expression or activity in a cell or bodily fluid, comprising
administering a vector that carries a gene encoding one or more
peptides selected from the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives and variants thereof, which peptides are
expressed in an amount that stimulates the animal's immune system
to produce antibodies that recognize and bind to the respective
peptide epitopes on the alpha subunit of the sodium potassium
ATPase thereby increasing the activity of the sodium potassium
ATPase in the cell or bodily fluid thereby treating or preventing
the disease.
33. The method of claim 32, wherein the disease is a member
selected from the group comprising diabetes, lung diseases, liver
diseases, urinary tract diseases, hemorrhagic shock,
gastrointestinal diseases including colitis, cataracts,
hypertension, Alzheimer's disease, eye disease, heart disease,
aging, cancer, kidney diseases, obesity and diseases of the nervous
system.
34. The method of claim 32, wherein the cell is a member selected
from the group comprising hepatocytes, kidney cells, red blood
cells, endothelial cells, lung cells, nerve cells, lens cells,
brain cells, muscle cells, cardiac myocytes, and any cells that
express sodium potassium ATPase.
35. The method of claim 32, wherein the vector further comprises
tissue specific promoters.
36. The method of claim 32 wherein the vector encodes more than one
peptide, or fragment, variant or derivative thereof.
37. A purified peptide comprising the amino acid sequence
HLLGIRETWDDRWIN.
38. An isolated antibody or antibody fragment that recognizes and
binds to an epitope comprising an amino acid sequence
HLLGIRETWDDRWIN or fragment, derivative or variant thereof in the
alpha subunit of any isoform of sodium potassium ATPase.
39. The antibody or antibody fragment of claim 38, wherein binding
of the antibody or antibody fragment to the alpha subunit of the
sodium potassium ATPase increases myocyte intracellular diastolic
and systolic calcium.
40. The antibody or antibody fragment of claim 38, wherein binding
of the antibody or antibody fragment to the alpha subunit of the
sodium potassium ATPase exerts a positive inotropic effect in
cardiac myocytes.
41. The antibody of claim 38, wherein the antibody is a polyclonal
antibody.
42. The antibody of claim 38, wherein the antibody is a monoclonal
antibody.
43. The antibody of claim 38, wherein the antibody is a humanized
antibody.
44. The antibody of claim 38, wherein the antibody is a humanized
antibody.
45. The antibody of claim 38, wherein the antibody is a SSA401.
46. A method for treating or preventing heart disease and/or muscle
contractile disorder, comprising administering to a patient in need
thereof an antibody, antibody fragment or small molecule that
recognizes and binds to an amino acid sequence comprising
HLLGIRETWDDRWIN or to a fragment derivative or variant thereof in
the alpha subunit of any isoform of the sodium potassium ATPase
enzyme, in an amount that increases sodium potassium ATPase
activity in myocytes thereby treating or preventing the heart
disease and/or muscle contractile disorder.
47. The method of claim 46, wherein the antibody is administered
systemically.
48. The method of claim 46, wherein the antibody is administered
locally.
49. A method for identifying compounds that activate sodium
potassium ATPase, the method comprising: a) identifying an assay
for quantifying sodium potassium ATPase activity, b) using the
assay, determining a baseline level of sodium potassium ATPase
activity in a control sample, c) in a separate binding assay,
incubating a compound of interest with a peptide having an amino
acid sequence selected from the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives and variants thereof under conditions that
permit the compound to bind to the peptide, d) determining whether
the compound binds to the peptide in step c), e) if the compound
binds to the peptide, then forming a mixture of the compound and
sodium potassium ATPase under conditions that permit the compound
to bind to the ATPase, f) using the assay of step a) and under the
same conditions as in step b) determining the level of ATPase
activity in the mixture, and g) if the level of ATPase activity in
the mixture is increased above the baseline level in the control
sample determined in step b), then concluding that the compound of
interest activates sodium potassium ATPase.
50. A method for increasing sodium potassium ATPase activity in a
cell, comprising contacting the cell with one or more antibodies or
antibody fragments, selected from the group comprising antibodies
and antibody fragments that recognize and bind to an amino acid
sequence in the alpha subunit of ATPase, which amino acid sequence
is selected from the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives and variants thereof.
51. A method for diagnosing heart failure and/or contractile
disorders in a patient comprising: a) isolating control heart
tissue from a healthy heart and heart tissue from the patient, b)
applying to the control and patient tissue an antibody, an antibody
fragment or a small molecule that recognizes and binds to the
epitope HLLGIRETWDDRWIN, or to fragments, derivatives and variants
thereof on any isoform of sodium potassium ATPase, and allowing
time for the antibody, antibody fragment or small molecule to bind
to the epitope, c) measuring intracellular diastolic and systolic
calcium and cell shortening of the control and patient heart tissue
to determine the ionotropic effect of the antibody, antibody
fragments or small molecule, and d) determining that the patient
has heart disease or a contractile disorder if the inotropic effect
on the patient heart tissue is lower than the ionotropic effect on
the control.
52. A sodium potassium ATPase activation site in the alpha subunit
selected from the group comprising the amino acid sequence
DVEDSYGQQWTYEQR, RSATEEEPPNDD, KRQPRNPKTDKLVNE, VPAISLAYEQAESD and
HLLGIRETWDDRWIN, and fragments, derivatives or variants thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of Provisional Application.
60/669,479, filed Apr. 8, 2005, the entire contents of which are
hereby incorporated by reference as if fully set forth herein,
under 35 U.S.C. .sctn.119(e).
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to antibodies, pharmaceutical
compositions and methods for increasing all isoforms of sodium
potassium ATPase (NKA) activity in a cell, tissue, organ, or bodily
fluid. It also relates to the use of the antibodies and
pharmaceutical compositions to treat or prevent diseases associated
with low NKA activity in an animal.
[0005] 2. Description of the Related Art
[0006] Sodium potassium ATPase (hereafter referred to as "NKA") [1]
is an integral membrane protein that couples the hydrolysis of ATP
to the vectorial transport of Na.sup.+ ions and K.sup.+ ions across
the plasma membrane of all animal cells [2]. The overall
stoichiometry of the reaction is three Na.sup.+ ions transported
out of the cell and two K.sup.+ ions into the cell for each ATP
hydrolyzed. It has been demonstrated that intact NKA is composed of
two subunits and only the .alpha.-subunit (.about.113 kDa) is
responsible for the catalytic activity of the enzyme. The smaller
.beta.-subunit (.about.35 kDa glycoprotein) is necessary for the
folding of the complex [3]. Recent transmembrane investigations
have suggested that the catalytic .alpha.-subunit traverses the
membrane ten times and both the N- and C-terminals of the
.alpha.-subunit are located on the cytoplasmic side [4, 5]. The
.beta.-subunit contains one hydrophobic region and only the
N-terminal is located on the cytoplasmic side [5, 6]. Several
isoforms of both .alpha.- and .beta.-subunits have been identified
[7]. There are two .alpha. isoforms (.alpha.1 and .alpha.2) on NKA
in rodent heart [8, 9] and three .alpha. isoforms (.alpha.1,
.alpha.2, and .alpha.3) in human heart [10, 11]. All isoforms of
NKA share the same catalytic function.
[0007] Although extensive studies have been made towards
understanding the structure/function relationship of NKA and its
central role in biology and medicine, little is known about the
activation of NKA and its biological influences. Enzymatic activity
of NKA is essential to living cells and continuity of life.
However, in certain diseases including heart diseases, liver
diseases, lung diseases, Alzheimer's disease, nervous system
diseases, intestinal diseases, cataracts and blood diseases, NKA
activity is depressed. In the fifty years since the discovery of
NKA, there have been no drugs or compounds identified that
selectively activate NKA activity.
[0008] The past approaches described in this section could be
pursued, but are not necessarily approaches that have been
previously conceived or pursued. Therefore, unless otherwise
indicated herein, the approaches described in this section are not
to be considered prior art to the claims in this application merely
due to the presence of these approaches in this background
section.
SUMMARY OF THE INVENTION
[0009] Specific drug interaction sites on NKA are provided for
increasing enzyme catalytic activity for prevention and treatment
of various diseases associated with low expression and activity.
There has never before been a report of precise activation sites or
drug interaction sites on NKA. Certain embodiments of the invention
are therefore directed to an NKA activation site in the alpha
subunit that is selected from the group comprising the amino acid
sequences DVEDSYGQQWTYEQR, RSATEEEPPNDD, KRQPRNPKTDKLVNE,
VPAISLAYEQAESD and HLLGIRETWDDRWIN, and variants, derivatives or
fragments thereof. Certain embodiments of the invention are also
directed to methods and compounds that interact with these
activation sites to treat or prevent diseases.
[0010] In one set of embodiments the invention is directed to
pharmaceutical compositions that increase NKA in a cell or bodily
fluid, such as hepatocytes, kidney cells, red blood cells,
endothelial cells, lung cells, nerve cells, lens cells, brain
cells, muscle cells, and cardiac myocytes. These compositions
contain an antibody, antibody fragment or small molecule that binds
to an activation site on the alpha subunit of any isoform of sodium
potassium ATPase (NKA), which activation site includes sites with
the amino acid sequences DVEDSYGQQWTYEQR, RSATEEEPPNDD,
KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN, and variants
or fragments thereof. In certain embodiments the antibody is one or
more of the following: SSA78, SSA95, SSA97, SSA401 and SSA412. Some
embodiments are directed to administering these pharmaceutical
compositions for treating or preventing a disease associated with
low sodium-potassium ATPase expression or activity in an animal
cell or bodily fluid, especially diseases like diabetes, lung
diseases, liver diseases, hypertension, urinary tract diseases,
hemorrhagic shock, gastrointestinal diseases including colitis,
cataracts, obesity, cancer, kidney disease, hypertension,
Alzheimer's disease, eye disease, heart disease, aging and diseases
of the nervous system.
[0011] One embodiment is directed to a newly discovered isolated
antibody SSA401 or a fragment thereof that recognizes and binds to
an epitope in the alpha subunit of any isoform of sodium potassium
ATPase having the amino acid sequence HLLGIRETWDDRWIN or to a
fragment, derivative or variant thereof. Such antibodies increase
myocyte intracellular diastolic and systolic calcium and exert a
positive inotropic effect in cardiac myocytes. Thus certain
embodiments are directed to the use of such antibodies or antibody
fragments to treat or prevent heart disease or muscle contractile
disorders. All of the antibodies described and used in the present
invention can be exogenous or endogenous, polyclonal, monoclonal or
humanized, and they can be administered systemically or
locally.
[0012] Some embodiments are directed to methods for increasing the
activity of any isoform of sodium-potassium ATPase in an animal
cell or bodily fluid, by administering to the animal an antibody,
an antibody fragment or a small molecule that recognizes and binds
to an epitope in the alpha subunit of any isoform of
sodium-potassium ATPase, which epitope has an amino acid sequence
that is a member selected from the group comprising
DVEDSYGQQWTYEQR, VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD,
HLLGIRETWDDRWIN, and fragments, derivatives and variants
thereof.
[0013] Certain other embodiments are directed to methods for
treating or preventing a disease in an animal that is associated
with low expression or activity of any isoform of sodium-potassium
ATPase in a cell or bodily fluid, by administering to the animal
one or more peptides representing antigenic sites or epitopes on
NKA from the group comprising DVEDSYGQQWTYEQR, VPAISLAYEQAESD,
KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and fragments,
derivatives and variants thereof, which peptides are administered
in an amount that stimulates the animal's immune system to produce
(endogenous) antibodies that recognize and bind to the respective
peptide epitopes (or antigenic sites) on the alpha subunit of the
NKA, thereby increasing ATPase activity in the cell or bodily
fluid. Some embodiments are directed to methods for increasing the
activity of any isoform of sodium potassium ATPase in an animal
cell or bodily fluid or to treating or preventing a disease
associated with low expression or activity of NKA, by administering
a vector that carries a gene encoding one or more peptides selected
from the activation sites in the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives and variants thereof. In some embodiments
the vector has tissue specific promoters.
[0014] Still other embodiments are directed to a purified peptide
that includes the amino acid sequence HLLGIRETWDDRWIN or fragments,
derivatives and variants thereof, and to antibodies, antibody
fragments and small molecules that recognize and bind to this
epitope on any isoform of NKA in any cell or tissue or bodily
fluid. Some embodiments are directed to methods whereby the
administration of such to antibodies, antibody fragments and small
molecules increases cardiac contraction and diastolic and systolic
calcium, and thus can be used to treat or prevent heart disease and
contractile disorders.
[0015] One embodiment is directed to a method for determining if a
compound activates sodium potassium ATPase. The method has the
steps of a) identifying an assay for quantifying sodium potassium
ATPase activity, b) using the assay, determining a baseline level
of sodium potassium ATPase activity in a control sample, c) in a
separate binding assay, incubating the compound with a peptide that
defines an antigenic site on the ATPase which peptide has an amino
acid sequence selected from the group comprising DVEDSYGQQWTYEQR,
VPAISLAYEQAESD, KRQPRNPKTDKLVNE, RSATEEEPPNDD, HLLGIRETWDDRWIN, and
fragments, derivatives or variants thereof under conditions that
permit the compound to bind to the peptide, d) determining whether
the compound binds to the peptide in step c), e) if the compound
binds to the peptide, then forming a mixture of the compound and
sodium potassium ATPase under conditions that permit the compound
to bind to the ATPase, f) using the assay of step a) and under the
same conditions as in step b) determining the level of ATPase
activity in the mixture, and g) if the level of ATPase activity in
the mixture is increased above the baseline level in the control
sample determined in step b), then concluding that the compound
activates sodium potassium ATPase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The present invention is illustrated by way of example, and
not by way of limitation, in the figures of the accompanying
drawings and in which like reference numerals refer to similar
elements and in which:
[0017] FIG. 1A shows that the primary amino acid sequence of the
D-R region (SSA412 binding site) is identical for the
.alpha.1-subunits of rat, human, dog, sheep, and pig. The D-R
region is also highly conserved in .alpha.2 (87%) and .alpha.3
(93%) NKA isoforms. FIG. 1B shows the results of
immunoprecipitation followed by electrophoresis and Western
blotting of whole rat heart cell lysates. For SDS gel: FIGS. 1B-A:
30 .mu.g of purified dog kidney NKA, FIGS. 1B-B: 10 .mu.g of
immunoprecipitates, FIGS. 1B-C: cell lysates. For Western blots:
FIGS. 1B-D: dog kidney NKA stained with SSA78 as positive control,
FIGS. 1B-E: 10 .mu.g immunoprecipitates stained with SSA412, FIGS.
1B-F: 10 .mu.g immunoprecipitates stained with SERCA2 as negative
control, FIGS. 1B-G 10 .mu.g immunoprecipitates stained with
secondary antibody as background control, FIGS. 1B-H: 10 .mu.g
immunoprecipitates stained with anti-.alpha.2 antibody, FIGS. 1B-I:
10 .mu.g immunoprecipitates stained with anti-.alpha.3 antibody,
FIGS. 1B-J: 10 .mu.g rat brain immunoprecipitates stained with
anti-.alpha.3 antibody, FIGS. 1B-K: rat cardiac SR vesicles stained
with SERCA2, FIGS. 1B-L: SR vesicles stained with SSA412. The
55,000 Da band in Western blots FIGS. 1B-E-J shows the denatured
heavy domains of IgG. SSA412 recognizes the .alpha.-subunit of
three isoforms of NKA, but not SR Ca.sup.2+-ATPase. Each of the
data represents one of six similar experiments.
[0018] FIG. 2A and FIG. 2B show the time course of activation of
purified rat (FIG. 2A: 0.125 mg/ml, ouabain-resistant) or dog (FIG.
2B: 0.125 mg/ml, ouabain-sensitive) NKA incubated with SSA412 (0.5
and 1.0 .mu.M) or with 1.0 .mu.M normal rabbit total IgG at
4.degree. C., or at room temperature FIG. 2C and FIG. 2D. NKA
activity was monitored at 0, 10, 30, 60, and 80 min time point for
experimental conditions in A and B, and 0, 10, 30, 60, 90, 120 min
for conditions in FIG. 2C and FIG. 2D.
[0019] FIG. 3A and FIG. 3B show that NKA activity is a function of
the concentration of SSA412: Purified dog (FIG. 3B, 1.25 .mu.g/ml)
and rat (FIG. 3A, 6.25 .mu.g/ml) NKA.
[0020] FIG. 3C and FIG. 3D show that in the presence of different
concentrations of MgATP, with or without 0.5 .mu.M of SSA412, the
Km values for both rat (FIG. 3C) and dog (FIG. 3D) NKA decreased 16
and 15% respectively, while the enzyme turnover rates increased.
FIG. 3C: For rat NKA control sample: Km.sub.MgATP=0.50 mM is
indicated by closed circles; open circles indicates rat NKA with
SSA412: Km.sub.MgATP=0.42 mM. FIG. 3D: For dog NKA control:
Km.sub.MgATP=0.53 mM is indicated by closed circles; open circles
indicates dog NKA with SSA412: Km.sub.MgATP=0.45 mM.
[0021] FIG. 3E and FIG. 3F show the effect of SSA412 on
phosphorylation of NKA: Purified rat (FIG. 3E) or dog (FIG. 3F) NKA
(17 nM) was phosphorylated in the presence of 100 mM Sodium or 20
mM K, 10 .mu.M MgATP, and 1 nM [.gamma.-.sup.32P] ATP with or
without SSA412. For both FIG. 3E and FIG. 3F black circles indicate
control: enzyme with sodium; open circles are with sodium plus 1
.mu.M SSA412; closed triangles are with potassium plus 1 .mu.M
SSA412, open triangles are with SODIUM plus 1 .mu.M rabbit IgG, and
closed triangles are without enzyme plus sodium plus 1 .mu.M
SSA412.
[0022] FIG. 3G and FIG. 3H show that binding of SSA412 causes no
changes in the apparent Na.sup.+:K.sup.+ affinity ratio for the
enzyme function, while accelerating NKA turnover rate. Each data
point represents a mean of four to five experiments. For both FIG.
3G and FIG. 3H, closed circles are controls and open circles are
plus 0.5 .mu.M SSA412.
[0023] FIG. 4A and FIG. 4B show representative illustrations of the
effect of SSA412 on Ca.sup.2+ transient amplitude (top trace) and
contraction (bottom trace) of Indo-1/AM-loaded rat heart cells
(FIG. 4A) before and after administration of SSA412 (0.25 .mu.M),
and (FIG. 4B) before and after administration of SSA412+0.5 mM
peptide blocker. FIG. 4C shows the average changes of cell
contraction for FIG. 4A and FIG. 4B. FIG. 4D shows the average
changes of Ca.sup.2+ transient for FIG. 4A and FIG. 4B. Data are
presented as % of control based on 20 independent measurements.
PB=peptide blocker.
[0024] FIG. 5 shows the immunofluorescent staining of SSA412 in rat
cardiac myocytes with SSA412 and without ouabain: FIG. 5A: a group
of cells at a magnification of 400.times., FIG. 5B: a single
myocyte at 1200.times., FIG. 5C: with SSA412 and peptide blocker,
FIG. 5D: secondary antibody control. With both ouabain and SSA412:
FIG. 5E: a group of cells with 5 mM ouabain at a magnification of
400.times., FIG. 5F: a single cell with 5 mM ouabain at
1200.times., FIG. 5G: cells with 10 mM ouabain, FIG. 5H: cells with
20 mM ouabain. The results show that the D-R region (SSA412 binding
site) is not an ouabain or digitalis interaction site. Each of
these data represents one of 12 similar immunofluorescent
stainings.
[0025] FIG. 6 shows [.sup.3H]ouabain binding to rat cardiac
sarcolemma (SL) membrane vesicles: FIG. 6A: control, FIG. 6B:
control+2 mM nonradioactive ouabain, FIG. 6C: with 1 .mu.M SSA412
SSA412, FIG. 6D: SSA412+2 mM nonradioactive ouabain, FIG. 6E: with
1 .mu.M SSA78, FIG. 6F: SSA78+2 mM nonradioactive ouabain. Specific
ouabain binding is presented as the difference between
[.sup.3H]ouabain binding in the presence and absence of
nonradioactive ouabain. The results represent mean values of 9
independent experiments each.
[0026] FIG. 7 shows that antibodies SSA95, SSA97, and SSA78 bind to
the alpha 1-subunit of NKA and increase ouabain-sensitive dog NKA
activity FIG. 7A: the control NKA activity level, FIG. 7B: with
SSA95, FIG. 7C: with SSA95+peptide blocker PB95 that has the amino
acid sequence KRQPRNPKTDKLVNE, FIG. 7D: with SSA78, FIG. 7E: with
SSA78+PB78 that has the amino acid sequence the RSATEEEPPNDD, FIG.
7F: with SSA97, and FIG. 7G: with SSA97+PB97 that has the amino
acid sequence VPAISLAYEQAESD.
[0027] FIG. 8 shows the effect of SSA412 on whole-cell L-type
calcium channel (LTCC) current of isolated rat myocytes. FIG. 8A:
time course of peak I.sub.Ca before and after administration of 1
.mu.M SSA412, where the currents were elicited every 10 sec. FIG.
8B: representative current traces recorded at -20 mV. C:
current-voltage relations I-V curves) before and after antibody
application. The results indicate that activation of NKA increases
LTCC function. Each of the data represents one of ten similar
results.
[0028] FIG. 9 shows the mechanism of activation of NKA-induced
increasing of Ca.sup.2+ transients. Rat neonatal ventricular
myocytes and `whole-cell` patch-clamp were used for the
investigation. Myoplasmic Ca.sup.2+ was determined from
simultaneous confocal optical recording of fluo-4 fluorescence. The
left-hand column represents a control myocyte, while the right-hand
column shows results from a myocyte previously exposed to SSA412
antibody (1.0 uM). FIG. 9A: the voltage pulse protocol, V.sub.M. A
ramp preceded each test pulse (-10 mV, 100 ms) in order to suppress
Na.sup.+ and T-type Ca.sup.2+ currents. FIG. 9B: LTCC currents
(I.sub.Ca), corrected for linear capacitance and leak using a P/4
protocol. FIG. 9C: myoplasmic Ca.sup.2+ during and after the test
pulse. Panel shows the myoplasmic Ca.sup.2+ as determined from
confocal line-scan (x vs. t) images. FIG. 9D: averaged results of
peak I.sub.Ca and peak myoplasmic Ca.sup.2+ elevation in each
group. The results show that the binding of SSA412 to NKA activates
LTCC activity, which simultaneously triggers SR calcium release.
The data suggest that 1) SR calcium is involved in the mechanistic
pathways of activation of NKA-mediated cardiac contraction, 2)
LTCC-regulated Ca.sup.2+-induced Ca.sup.2+-release (CICR) may
account for how activation of NKA triggers the positive inotropic
effect. Values represent the mean (.+-.SEM). Values represent the
mean (.+-.SEM).
[0029] FIG. 10 shows the time course of the effect of .alpha.- and
.beta.-adrenergic receptor inhibitors on activation of the
NKA-induced positive inotropic effect in isolated rat myocytes.
Cell contractions were measured in the presence or absence of
SSA412 (0.5 .mu.M) with or without inhibitors. Black circles: with
1 .mu.M isoproterenol. Open circles: with 1 .mu.M
isoproterenol+both prazosin and propennol (7 .mu.M each). Black
triangles: with SSA412 in the presence of both prazosin and
propennol (7 .mu.M each). Data are presented as percent of control
(n=8 cells for each group). Inhibitors did not affect the
biological effect of SSA412 while completely abolishing the
activity of agonist isoproterenol.
[0030] FIG. 11 shows that activation of NKA has no effect on cyclic
AMP (cAMP) formation in rat cardiac myocytes. Isolated rat cardiac
myocytes were treated with phosphodiesterase inhibitor,
3-isobutyl-1-methylxanthine (IBMX, 1 mM) or DMSO side by side for
30 min at room temperature prior to being incubated with either
active SSA412 antibody or boiled SSA412 (4 .mu.M each) for 30 min,
or with b-AR agonist, isoproterenol (1 mM) for 5 min as a positive
control. Formation of cAMP was assayed using .sup.3H-cAMP. Protein
concentration was measured using bovine serum albumin as standard.
Each of the data represents the mean of three independent
experiments.
[0031] FIG. 12 shows representative results of the time course of
rat heart cell contraction with or without SSA401. Time runs from
left to right. Control baseline of rat heart cell contractions are
shown at zero min. Increased cell contraction as shown at 15 and 30
min following administration of SSA401.
[0032] FIG. 13 shows that activation of NKA is a function of the
concentration of SSA412. Purified rat NKA (6.25 .mu.g/ml) was
incubated with different concentrations of SSA401 (as indicated in
the figure) for 60 min at 4.degree. C. prior to enzyme assay.
DETAILED DESCRIPTION
[0033] In the following description, for the purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the present invention. It will
be apparent, however, to one skilled in the art that the present
invention may be practiced without these specific details.
Definitions for certain terms used herein are set forth below at
the end of the detailed description.
[0034] It has been discovered that certain antibodies that bind to
specific sites on the alpha subunit of NKA dramatically increase
NKA activity. "NKA" and "sodium potassium ATPase" are used
interchangeably herein. These specific sites are called activation
sites. There has never before been a report of precise activation
sites or drug interaction sites on NKA. We have discovered that the
amino acid sequences DVEDSYGQQWTYEQR, RSATEEEPPNDD,
KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN are activation
sites on the alpha subunit of NKA. Certain embodiments of the
invention are therefore directed to these activation sites and to
fragments, derivatives or variants thereof. Antibodies and
fragments thereof that bind to the alpha subunit thereby increasing
NKA activity are collectively referred to herein as "NKA
activity-increasing antibodies." The NKA-activity-increasing
antibodies all bind to various epitopes or antigenic determinants
defined by the above amino acid sequences or fragments, derivatives
and variants thereof on any isoforms of NKA. They include:
[0035] 1) Those that bind to the amino acid sequence
DVEDSYGQQWTYEQR in the H7-H8 domain (including the SS412 antibody
referred to also as the KX-1 antibody), or to fragments,
derivatives and variants thereof.
[0036] 2) Those that bind to the amino acid sequence RSATEEEPPNDD
in the H1-H2 domain (including the SSA78 antibody also referred to
as the Jianye 2 antibody), or to fragments, derivatives and
variants thereof.
[0037] 3) Those that bind to the amino acid sequence
KRQPRNPKTDKLVNE in the H5-H6 domain (including the SSA95 antibody
also referred to as the Jianye antibody), or to fragments,
derivatives and variants thereof.
[0038] 4) Those that bind to the amino acid sequence VPAISLAYEQAESD
in the H5-H6 domain (including the SSA97 antibody also referred to
as the Zulan antibody), or to fragments, derivatives and variants
thereof.
[0039] 5) Those that bind to the newly discovered amino acid
sequence HLLGIRETWDDRWIN in the H7-H8 domain of NKA adjacent to the
D-R region (including the newly discovered SSA401 or KX-2
antibody), or to fragments, derivatives and variants thereof. The
HLLGIRETWDDRWIN site on NKA is a newly discovered site capable of
causing both NKA activation and a positive ionotropic effect on
cardiac myocytes.
[0040] All isoforms of the alpha subunit of NKA share the same
enzymatic function. Therefore any antibody or antibody fragment or
small molecule that binds to any of the five activation sites
described above or to a fragment, derivative or variant of the
activation site on any of the isoforms of NKA will increase the
enzymatic activity. Certain embodiments of the present invention
are directed to pharmaceutical compositions that increase
sodium-potassium ATPase activity in an animal cell or bodily fluid,
which compositions include any antibody, antibody fragment or small
molecule that binds to an activation site on NKA, which activation
sites include DVEDSYGQQWTYEQR, RSATEEEPPNDD, KRQPRNPKTDKLVNE,
VPAISLAYEQAESD and HLLGIRETWDDRWIN, and variants or fragments
thereof. Other embodiments are directed to methods of increasing
sodium-potassium ATPase activity in an animal cell or bodily fluid,
by administering to the animal an NKA activity-increasing amount of
one or more of the five NKA activity-increasing antibodies listed
above. Antibodies useful in the present invention can be monoclonal
or polyclonal, and may be humanized. Animal cells include mammalian
cells, preferably human cells, and include but are not limited to
liver cells, heart cells, kidney cells, nerve cells, red blood
cells, endothelial cells, brain tissue, lung cells, lens cells and
cells in the gastrointestinal tract. Any cell that expresses NKA
can be contacted with the NKA activity-increasing antibodies to
increase NKA activity in vivo or in vitro. Antibodies can be
administered to an animal in any way known in the art, including
locally and systemically.
[0041] The invention also includes increasing NKA activity in an
animal cell by administering a vaccine that includes one or more
peptides selected from a group of peptides having the amino acid
sequences DVEDSYGQQWTYEQR, RSATEEEPPNDD, KRQPRNPKTDKLVNE,
VPAISLAYEQAESD and HLLGIRETWDDRWIN, and variants or fragments
thereof. These peptides represent antigenic determinants or
epitopes on the alpha 1-subunit of NKA. The peptide vaccines
stimulate the host immune system to generate antibodies against the
respective one or more peptide epitopes. The results presented
herein show that the in vivo-generated antibodies bind to the
respective epitope/antigenic determinant on the alpha 1-subunit of
NKA thereby increasing NKA activity. Methods for making, isolating
and purifying NKA activity-increasing antibodies and the
above-identified peptide antigenic determinants are described in
U.S. Patent Applications 20040057956 and 20030228315, the entire
contents of which are hereby incorporated by reference as if fully
set forth herein.
[0042] NKA activity is known to be low in aging and certain
diseases, such as Alzheimer's disease [32-34], aging [35], kidney
diseases and hypertension [36-38], obesity [39], insulin resistance
and complications [40-45], diabetes [46, 61], lung diseases [47],
colitis [48], Rye's syndrome [49], liver diseases [50], urinary
tract diseases [52], intestinal diseases [53-54], hemorrhagic shock
[55], cataracts [56-57], and other diseases of the nervous system
[58-60] or heart associated with low levels of NKA activity. The
NKA pump has been well studied for its role in the regulation of
ion homeostasis in mammalian cells. Recent studies suggest it might
have multiple functions such as a role in the regulation of tight
junction structure and function, induction of polarity, regulation
of actin dynamics, control of cell movement, and cell signaling.
These functions appear to be modulated by NKA activity as well as
protein-protein interactions of the alpha and beta subunits. A
reduction or impairment of NKA function or reduced subunit
expression levels have been implicated in kidney diseases such as
cancer, tubulointerstitial fibrosis, and ischemic nephropathy. [50]
Until the discovery of the active sites on NKA and of NKA
activity-increasing antibodies as described herein, there was no
known agent that could be used to prevent or treat such diseases by
stimulating NKA activity. Therefore some embodiments of the present
invention are directed to methods for preventing or treating
diseases associated with decreased NKA activity by administering to
a diseased animal one or more NKA activity-increasing antibodies or
antibody fragments or a small molecule that binds to an active site
on NKA, in an amount that increases sodium-potassium ATPase
activity in the appropriate cells or tissues. In some embodiments
the one or more NKA activity-increasing antibodies are administered
systemically, for example to increase NKA activity in red blood
cells or endothelium, or locally for example to treat
cataracts.
[0043] In another embodiment, the invention provides a method for
increasing NKA activity by administering to an animal a vector
carrying a gene that encodes one or more of the five amino acid
sequence epitopes on NKA (DVEDSYGQQWTYEQR, RSATEEEPPNDD,
KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN, and variants,
derivatives or fragments thereof. The vectors can be made using any
technique known in the art of recombinant DNA or RNA technology.
The in vivo expression of the antigenic determinants or epitopes
acts like a vaccine causing the host immune system to generate the
respective one or more NKA activity-increasing antibodies. In
preferred embodiments, these vectors are under the control of
tissue specific promoters so that the vectors can be optimally
expressed in the targeted cell or bodily fluid. Such tissue
specific promoters come from tissues including pancreas,
endothelium, epithelium, kidney, liver, bladder, intestine, colon,
brain, neurons, the gastrointestinal tract, heart, lung, muscle,
and nervous system. These vectors can also be used to generate sera
comprising the NKA activity-increasing antibodies using standard
methods such as immunizing mammals.
[0044] Some embodiments of the present invention are directed to
the new antibody SSA401; to pharmaceutical compositions that
contain the antibody or a fragment thereof, and to its therapeutic
use to treat diseases associated with low NKA activity, including
heart diseases and contractile disorders. This antibody, like the
others, can be monoclonal or polyclonal and humanized. Another
embodiment is directed to the peptide HLLGIRETWDDRWIN, and to its
use as a diagnostic agent for treating or preventing heart disease
or other contractile disorders, by detecting, in standard assays,
such as ELISAs, RIAs and the like, peptides which are indicative of
contractile disorders. In another preferred embodiment, the
invention provides for pharmaceutical compositions comprising the
HLLGIRETWDDRWIN peptide (or fragment, derivative or variant) for
treating or preventing heart diseases and contractile diseases.
The Alpha 1-Subunit is a Highly Conserved Antigenic Site in NKA
Isoforms
[0045] The functional NKA comprises a 113-kDa catalytic alpha
(.alpha.) subunit together with a noncatalytic 35-kDa beta (.beta.)
subunit and appears to exist as an heterodimer. NKA couples the
hydrolysis of one molecule of ATP to the outward translocation of
three sodium ions and inward translocation of two potassium ions
against their steep electrochemical gradients. Thus NKA maintains
the normally high-potassium and low-sodium concentrations in the
cytoplasm of animal cells. Kinetic experiments have indicated that
the catalytic alpha subunit can exist in at least four
conformations as it passes through each turnover. The four
conformations are E.sub.1, E.sub.1-P, E.sub.2-P, and E.sub.2. It is
thought that E.sub.1 is the inward-facing form of the enzyme, which
normally releases K.sup.+ as a product and receives Na.sup.+ as a
substrate. The E.sub.1 conformation has high affinity for ATP and
participates in sodium-dependent phosphorylation to form E.sub.1-P.
The E.sub.1-P conformation is the one in which the protein has
surrounded the three Na.sup.+ ions on their way to the outside. The
E.sub.2-P conformation is the outward-facing form and releases the
three Na.sup.+ ions as products and receives the two K.sup.+ ions
as substrates. Upon dephosphorylation, the E.sub.2 conformation is
formed. This conformation surrounds the two K.sup.+ ions and has
low affinity for ATP. It is during the transitions among these four
forms that the cations traverse the plasma membrane. All isoforms
of the alpha subunit of NKA share the same enzymatic function.
Therefore any antibody or small molecule that binds to any of the
five activation sites described herein or to a fragment, derivative
or variant of the activation site of any of the isoforms of NKA
increases the enzymatic activity.
[0046] NKA regulates both excitability and contractility of the
heart. Previous studies by our laboratory identified the SSA 412,
78, 95 and 97 antibodies that recognize specific sites in the
extracellular domain of the alpha subunit (also referred to as
".alpha.1") rat NKA. [12, 31-32], the entire contents of which is
incorporated by reference as if fully set forth herein. The SSA412
antibody (also referred to as the KX-1 antibody) and the SS78
antibody (also referred to as the Jianye-2 antibody) are described
in detail in the US Patent Application No. 20040057956; the SSA 95
and SSA 97 antibodies are described in US Patent Application No.
20030228315, the entire contents of which is incorporated by
reference as if fully set forth herein. These four antibodies were
shown to have therapeutic use for treating or preventing heart
diseases and contractile disorders by producing a positive
ionotropic effect and increasing muscle contraction. A new antibody
SSA401 has now been discovered that posses these ionotropic
properties in addition to being able to increase NKA activity.
[0047] Most of the detailed experiments on NKA activity were done
using the site-specific antibody SSA412 made against the D-R region
[18, 19], which resides within the extracellular H7-H8 domain of
rat .alpha.1 NKA (FIG. 1A). Comparison of amino acid sequences
shows that the D-R region is highly conserved: it is identical for
.alpha.1 NKA in different species [3, 18-22], 87% identity between
.alpha.1 and .alpha.2 [18, 19, 23], and 93% for .alpha.3 [18, 19,
24] isoform (FIG. 1A). Therefore antibodies that work in rodents,
will work similarly in higher mammals. Using immunoprecipitation
and Western blotting, we examined the interaction between SSA412
and its antigenic site on NKA. SDS gel electrophoresis reveals that
SSA412 specifically binds to and precipitates NKA (FIGS. 1B-B) from
rat heart cell lysates (FIGS. 1B-C). This is confirmed by highly
purified dog NKA (FIGS. 1B-A) and a control antibody SSA78 (FIGS.
1B-D), which targets the H1-H2 domain of the enzyme [12]. Western
blots show that SSA412 and SSA78 recognize the .alpha.-subunits of
NKA (FIGS. 1B-E and FIGS. 1B-D). Since only high population
.alpha.1 and low .alpha.2 are expressed in rat heart, the results
of Area-Density Calculation (LabWorks analysis software,
Ultraviolet Products Bioimaging Inc., Upland, Calif., USA) show
that SSA412 immunoprecipitates 95% .alpha.1 (FIGS. 1B-E) and 5%
.alpha.2 (FIGS. 1B-H) from isolated rat cardiomyocytes. SSA412
co-precipitates .alpha.3 NKA from rat brain homogenates (FIGS.
1B-J). These data suggest that SSA412 interacts with its antigenic
site which is highly conserved in NKA isoforms. SERCA2 antibody
recognizes Ca.sup.2+-ATPase from SR vesicles (FIGS. 1B-K), but
SSA412 does not cross-react with SR Ca.sup.2+-ATPase (FIGS.
1B-L).
NKA is Activated by Antibodies that Bind to the Alpha 1-Subunit
[0048] NKA is a ubiquitous and critical enzyme for electrolyte
balance that was discovered a half century ago. [1] Although NKA
activity is depressed in many diseases, no one has found a means
for activating this enzyme until now. Previous studies in cardiac
myocytes showed that the binding of antibodies SSA412, SSA95,
SSA97, and SSA78 to extracellular sites in the alpha-subunit of NKA
produced various physiologic effects, i.e. the increase in calcium
transients and myocyte contraction. Cardiac glycosides that also
have positive ionotropic effects in cardiac myocytes were presumed
to work by inhibiting NKA. Here we show that the native catalytic
power of NKA is markedly elevated when antibody-enzyme interaction
occurs at various extracellular regions in the .alpha.-subunit of
the enzyme. The results of the experiments described herein show
that the binding of antibodies SSA412, SSA95, SSA97, SSA78 and SSA
401 to extracellular sites in the alpha 1-subunit of NKA activates
the enzyme. These antibodies also recognize the similar sites on
isoforms of NKA. Because the extracellular peptide regions of the
alpha 1-subunit of NKA to which the antibodies bind are highly
conserved among species and among tissues within a species, these
antibodies have wide therapeutic application to treat any disease
or metabolic imbalance in an animal that is associated with lower
than normal levels of NKA activity. Energy deficiency and
dysfunction of the NKA are common consequences of many
diseases.
[0049] Example 1 below describes how the activation of NKA induced
by various antibodies was determined. Example 2 describes
Immunoprecipitation, blotting, and immunofluorescent staining of
the antibodies. Example 3 describes the methods for measuring
physiologic indices of intracellular Ca.sup.2+ transients and cell
contraction in cardiac myocytes. Example 4 describes the Isolation
of sarcolemmal vesicles and the purification of NKA. Example 5
describes methods of measuring NKA activity, and Example 6
describes methods of measuring NKA phosphorylation.
[0050] A first series of experiments were conducted testing whether
antibody SSA412, binding to the D-R region of the alpha 1-subunit,
affects NKA activity. NKA activity was determined based on Jack
Kyte's method as previously described [17, which is hereby
incorporated by reference as if set forth fully herein]. The
results presented below show that NKA activity levels can be
increased by SSA412 antibody binding. FIG. 2A and FIG. 2B show the
time course of activation of purified rat (FIG. 2A: 0.125 mg/ml,
ouabain-resistant) or dog (FIG. 2B: 0.125 mg/ml, ouabain-sensitive)
NKA incubated with SSA412 (0.5 and 1.0 .mu.M) or with 1.0 .mu.M
normal rabbit total IgG at 4.degree. C. or at room temperature
(FIG. 2C and FIG. 2D). The control is indicated by closed circles,
0.5 mM SSA412 by open circles, 1.0 mM SSA412 by closed triangles
and 1.0 mM rabbit IgG by open triangles.
[0051] Different concentrations of SSA412 (0.5 or 1.0 .mu.M) were
incubated with purified ouabain resistant-(rat) and sensitive-(dog)
NKA prior to ATPase assay [17]. FIG. 2A and FIG. 2B show that
SSA412 markedly activated both rat and dog NKA function. The
catalytic turnover of rat NKA was 34, 36, 42, 53, and 60 s.sup.-1
following 0, 10, 30, 60, and 80 min incubation with 0.5 .mu.M
SSA412 at 4.degree. C., and 34, 37, 48, 61, 72 s.sup.-1 with 1.0
.mu.M SSA412 (FIG. 2A). Under the same experimental conditions as
for rat NKA, the turnover of ouabain-sensitive dog NKA also
increased. NKA in dog was 53, 54, 58, 77, 86 s.sup.-1 with 0.5
.mu.M SSA412, and 53, 55, 64, 85, 99 s.sup.-1 with 1.0 .mu.M SSA412
(FIG. 2B). By contrast, no significant changes in control samples
(without SSA412) and in the presence of 1.0 .mu.M total rabbit IgG
for both rat and dog enzymes (FIG. 2A and FIG. 2B) were found,
indicating the specificity of SSA412-induced activation of NKA.
These results suggest that NKA catalytic activity can be
significantly enhanced beyond normal levels by binding with SSA412
antibody, and that the D-R region is an effective site of NKA
activity.
[0052] This novel NKA activation was verified at room temperature
(RT) using the same SSA412 concentrations, except that SSA412 was
added to the samples after 60 min. In the absence of the enzyme
substrate MgATP, the activity of purified NKA is gradually
denatured at RT as a function of time as shown in FIG. 2C and FIG.
2D (black circles). When partially inactivated NKA was exposed to
SSA412, it remarkably protected both rat and dog enzyme functions
by enhancing NKA catalytic turnover against further denaturing: rat
NKA turnover was 16 s.sup.-1 (FIG. 2C, black circles) following 60
min at RT before exposure to SSA412, and increased to 23 and 29
s''.sup.1 after incubation with 0.5 .mu.M SSA412 for 30 and 60 min,
respectively (FIG. 2C, open circles). Under the same partially
inactivation conditions, rat NKA turnover was further accelerated
to 27 and 34 s.sup.-1 in the presence of 1.0 .mu.M SSA412 (FIG. 2C,
black triangles). A similar phenomenon was also observed for dog
NKA: enzyme catalytic turnover was 32 s.sup.-1 at RT for 60 min
(partial inhibition state at RT), and increased to 36 and 46
s.sup.-1 after exposure to 0.5 .mu.M SSA412 for 30 and 60 min, and
41 and 52 s.sup.-1 with 1.0 .mu.M SSA412 under the same
experimental conditions.
[0053] Activation of NKA was further examined by the determination
of the effective concentration of SSA412. Purified dog (1.25
.mu.g/ml) and rat (6.25 .mu.g/ml) NKA were incubated at the
different concentrations of SSA412 for 60 min prior to the standard
ATPase assay. Purified dog (FIG. 3A, 1.25 .mu.g/ml) and rat (FIG.
3B, 6.25 .mu.g/ml) NKA was used. Experimental results show that the
rate of activation of NKA is a function of the concentration of
SSA412. The maximum turnover of the enzyme is over 2 times faster
than that of the controls for both ouabain-resistant (FIG. 3A) and
ouabain-sensitive NKA (FIG. 3B). The half effective concentration
(EC.sub.50) of SSA412 is 0.14 .mu.M for rat and 0.15 .mu.M for dog
NKA. These data demonstrate that the D-R region is an important
effective site of NKA and that SSA412 is a novel effector to
stimulate NKA activity, in which interaction with the enzyme
elevates the NKA-catalyzed reaction.
Characteristics Of NKA Activation
[0054] Hydrolysis of MgATP is one of the fundamental components of
the catalytic function of NKA. The Michaelis constant (K.sub.m)
indicates the mode of regulating the activity of an enzyme. The
effect of SSA412 on the numerical value of K.sub.m of MgATP in the
activation of NKA was next determined. In the presence of 0.5 .mu.M
of SSA412 with different MgATP concentrations, the K.sub.m values
were lowered 16 and 15% for rat and dog NKA while maximum enzyme
activity (or enzyme turnover) increased in both cases (FIG. 3C and
FIG. 3D). Moreover, binding of SSA412 to NKA promotes the net
.sup.32P-phosphorylation labeling (FIG. 3E and FIG. 3F), confirming
that SSA412-activated NKA is based on the enhanced rate of MgATP
hydrolysis. No changes occurred in the .sup.32P-phosphorylation
labeling in the absence of SSA412 or presence of total rabbit
IgG.
[0055] NKA-catalyzed hydrolysis of MgATP is a function of Na.sup.+
plus K.sup.+ concentrations [25]. It was next determined whether
the binding of SSA412 to the D-R region of NKA would affect local
Na.sup.+/K.sup.+ binding concentrations and ion active transport.
No significant changes in the optimal binding concentrations of
Na.sup.+ and K.sup.+ were detected while NKA activity and
Na.sup.+/K.sup.+ ion transport were significantly increased (FIG.
3G and FIG. 3H, open circles). To confirm the SSA412-enhanced
Na.sup.+/K.sup.+ ion transport, we investigated the effect of
SSA412 on the initial rate of Na.sup.+ and K.sup.+ transport
separately. When the cytoplasmic site of the enzyme is saturated
with 100 mM Na.sup.+ and 3 mM MgATP, the initial rate of K.sup.+
transport increased two-fold with 5 mM K.sup.+ in the presence of 1
.mu.M SSA412 (Table 1). The same is true for Na.sup.+ transport,
SSA412 also doubled the initial rate of Na.sup.+ transport while
the extracellular site is saturated with 20 mM K.sup.+ and 3 mM
MgATP in the presence of 20 mM Na.sup.+ (Table 1). Condition 1:
[Na.sup.+]=100 mM, [K.sup.+]=5 mM. Condition 2: [Na.sup.+]=20 M,
[K.sup.+]=20 mM. In both conditions: [MgATP]=3 mM, SA412]=1
.mu.M.
TABLE-US-00001 TABLE 1 SSA412 ENHANCES THE APPARENT INITIAL RATE OF
SODIUM AND POTASSIUM TRANSPORT UNDER DIFFERENT EXPERIMENTAL
CONDITIONS (N = 5) Condition 2 Condition 1 Na.sup.+-regulated
K.sup.+-regulated initial rate initial rate Samples (.mu.mol Pi
mg.sup.-1 min.sup.-1) (.mu.mol Pi mg.sup.-1 min.sup.-1) Rat NKA
Control 1.82 .+-. 0.2 1.64 .+-. 0.1 Rat NKA + SSA412 4.03 .+-. 0.5
3.64 .+-. 0.4 Dog NKA Control 21.8 .+-. 1.6 14.5 .+-. 2.1 Dog NKA +
SSA412 45.7 .+-. 4.7 30.8 .+-. 3.3
Mechanisms Of Activation Of NKA-Mediated Biological Processes
[0056] It was known from previous work by our laboratory that
SSA412, SSA78, SSA95 and SSA97 elicit a positive ionotrophic
response in cardiac myocytes, but the mechanism of action was not
known. Here we show that when isolated rat myocyte is loaded with
the calcium indicator Indo-1/AM, binding of SSA412 (0.25 .mu.M) to
NKA increased both myocyte intracellular Ca.sup.2+ transient
amplitude (1.24-fold, n=20) (FIG. 4A upper trace) and contraction
(1.67-fold, n=20) (FIG. 4A lower trace). The increase in calcium
and contraction was blocked with a peptide blocker DVEDSYGQQWTYEQR.
These results reveal that activation of NKA generates a positive
inotropic effect and suggests that amino acids residing in the D-R
region may be vital residues to NKA activity, which plays an
important role in cardiac regulation. FIG. 4C shows the average
changes of cell contraction for FIG. 4A and FIG. 4B. FIG. 4D shows
the average changes of the Ca.sup.2+ transient for FIG. 4A and FIG.
4B. Data are presented as % of control based on 20 independent
measurements. PB=peptide blocker.
[0057] To understand the mechanisms underlying the biological
activity of these inotropic antibodies and activation of the
NKA-induced increase of calcium ion transients and muscle
contraction, we tested whether activation of NKA affects cardiac
L-type Ca.sup.2+-channel (LTCC) function. It was discovered that
cardiac LTCC activity is increased following exposure to antibody
SSA412 in isolated adult rat myocytes (FIGS. 8A, 8B and 8C). No
significant changes occurred in the absence of SSA412 (before
adding antibody). These data directly link LTCC activity to the
activation of NKA-regulated increase of [Ca.sup.2+].sub.i; and
cardiac contraction.
[0058] We next tested the functional relationship between
activation of NKA-induced enhancement of LTCC current and
sarcoplasmic reticulum (SR) calcium release in rat neonatal
myocytes. FIG. 9 shows that the binding of SSA412 to NKA activates
LTCC activity, which simultaneously triggers SR calcium release in
rat neonatal ventricular myocytes grown on cover slips and
voltage-clamped (`whole-cell` patch-clamp). The data suggest that
(a) SR calcium is involved in the mechanistic pathways of
activation of NKA-mediated cardiac contraction, and (b)
LTCC-regulated Ca.sup.2+-induced Ca.sup.2+-release (CICR) may
account for how activation of NKA triggers the positive inotropic
effect.
[0059] Adrenergic receptors play important role in regulating
cardiac contraction. To see whether the .alpha.- and
.beta.-adrenergic receptors are involved in the activation of
NKA-regulated LTCC activity, we looked at the time course of the
effect of .alpha.- and .beta.-adrenergic receptor inhibitors on
activation of the NKA-induced positive inotropic effect. FIG. 10
shows that neither the .alpha.-adrenergic receptor inhibitor
prazosin nor the .beta.-adrenergic receptor inhibitor propennol (7
.mu.M each) affected activation of the NKA-induced positive
inotropy, while both inhibitors eliminated action of the agonist
isoproterenol. The data demonstrate that activation of NKA-induced
biological action is independent of .alpha.- and .beta.-adrenergic
receptor-mediated pathways.
[0060] Cyclic adenosine monophosphate (cAMP) is a very important
molecule that controls many biological activities. We further
investigated whether cAMP-dependent pathways are involved in the
activation of NKA-regulated biological processes. FIG. 11 indicates
that activation of NKA has no effect on cAMP formation in isolated
rat cardiac myocytes, indicating that activation of NKA-regulated
LTCC activity is independent of cAMP signaling pathways.
SSA412 Binding Site is Not a Digitalis-Binding Site
[0061] NKA is a target receptor for digitalis and related cardiac
glycosides [26-29]. These drugs induce a positive inotropic effect
by inhibiting NKA activity [30]. To determine whether the newly
discovered effective site is one of the digitalis interacting-sites
in NKA, immunofluorescence microscopy was performed on rat heart
cells. FIG. 5A shows that SSA412 specifically binds to the D-R
region of NKA on the surface of the cell membrane (FIG. 5A and FIG.
5B). Ouabain, at 5, 10, and 20 mM, does not compete with SSA412
binding to the D-R region of NKA (FIG. 5E-H). To verify this
observation, a [.sup.3H]ouabain labeling experiment was performed.
Isolated cardiac sarcolammal membranes right-side-out vesicles were
incubated with [.sup.3H]ouabain in the presence or absence of
SSA412. The results show that SSA412 (1 .mu.M) did not decrease or
prevent [.sup.3H]ouabain binding to the enzyme. By contrast, the
SSA78 (1 .mu.M) made against the H1-H2 domain of the enzyme,
reduced the [.sup.3H]ouabain labeling by 33% (FIG. 6). These data
clearly demonstrate that the D-R region in the .alpha.-subunit of
NKA is not one of the ouabain interacting sites of the enzyme.
The SSA78, SSA95 and SSA97 Also Increase NKA Activity
[0062] To see whether there were other activation sites on NKA, the
effect of SSA78, SSA95 and SSA97 was tested. FIG. 7 shows that NKA
activity in dog, which is ouabain-sensitive, was also increased by
activation agents SSA78, SSA95, and SSA97 that bind to the alpha
1-subunit of NKA at amino acid sequences. The antibody-induced
increase in NKA by binding to each of these antibodies was blocked
with the respective peptide blockers PB78 (RSATEEEPPNDD), PB95
(KRQPRNPKTDKLVNE),) and PB97 (VPAISLAYEQAESD).
A New Antibody SSA401 that Binds to the Alpha 1-Subunit is
Discovered
[0063] We discovered a new antibody SSA401 (also referred to as the
KX-2 antibody) that binds to the alpha 1-subunit of NKA,
specifically to the epitope HLLGIRETWDDRWIN. FIG. 12 shows that the
SSA401 antibody strikingly enhanced the velocity of shortening and
the force of contraction of isolated rat heart cells. The result
indicates that the SSA401 antibody is a new inotropic agent.
[0064] FIG. 13 shows that the binding of SSA401 to purified NKA
activates enzyme function. The catalytic activity of rat NKA was
increased by 120.+-.7, 146.+-.9, 163.+-.4, 179.+-.5, 198.+-.5, and
210.+-.8% (compared with the control) in the presence of 0.1, 0.3,
0.5, 0.7, 1.0, and 2.0 .mu.M SSA401, respectively. The half
effective concentration (EC.sub.50) of SSA401 is approximately 0.4
.mu.M. The data indicate that the HLLGIRETWDDRWIN region is another
activation site of the enzyme and show that the mechanism
underlying SSA401 induced positive inotropic effect is based on the
activation of NKA. Therefore, an embodiment of the present
invention is directed to a new antibody SSA401 in monoclonal,
polyclonal and humanized forms. In a preferred embodiment, the
invention provides for the therapeutic use of antisera, polyclonal
and monoclonal and/or humanized SSA401 antibodies that specifically
bind to amino acid the HLLGIRETWDDRWIN sequence of the NKA enzyme,
for treating patients suffering from or susceptible to heart
disease and/or muscle contractile disorders, and for treating or
preventing any disease associated with low NKA activity. Certain
embodiments are also directed to both exogenous and endogenous
SSA401 antibody used to increase cardiac contraction for the
treatment of heart failure. This antibody also eliminates the
effects of certain precipitating drugs, including negative
inotropic agents (e.g., certain calcium channel blockers and
antiarrhythmic drugs like disopyramide), cardiotoxins (e.g.,
amphetamines) and plasma volume expanders (e.g., nonsteroidal
antiinflammatory agents and glucocorticoids).
[0065] Some embodiments of the present invention are directed to
methods for identifying compounds that activate sodium potassium
ATPase by binding to an activation site. One such method has the
steps of a) identifying an assay for quantifying sodium potassium
ATPase activity, b) using the assay, determining a baseline level
of sodium potassium ATPase activity in a control sample, c) in a
separate peptide binding assay, incubating a compound of interest
with a peptide having an amino acid sequence selected from the
group comprising DVEDSYGQQWTYEQR, VPAISLAYEQAESD, KRQPRNPKTDKLVNE,
RSATEEEPPNDD, HLLGIRETWDDRWIN, and fragments, derivatives and
variants thereof under conditions that permit the compound to bind
to the peptide, d) determining whether the compound binds to the
peptide in step c), e) if the compound binds to the peptide, then
forming a mixture of the compound and sodium potassium ATPase under
conditions that permit the compound to bind to the ATPase, f) using
the assay of step a) and the same conditions as step b) determining
the level of ATPase activity in the mixture, and g) if the level of
ATPase activity in the mixture is increased above the baseline
level in the control sample determined in step b), then concluding
that the compound activates sodium potassium ATPase.
[0066] The term "treatment" or grammatical equivalents encompasses
the improvement and/or reversal of the symptoms of a disease.
[0067] The term "individual" as used herein refers to vertebrates,
particularly members of the mammalian species and includes but is
not limited to, domestic animals, sports animals, primates and
humans; more particularly, the term refers to humans.
[0068] As used herein, "contractile disorders" refers to the
abnormal contractile response of muscle cells as compared to normal
muscle cells. Examples of such disorders are arhythmia,
tachyrhithmia, and the like.
[0069] Heart disease refers to any heart disease that is responsive
to treatment with one or more of the antibodies described
herein.
[0070] A "polynucleotide" refers to a polymeric form of nucleotides
of any length, either ribonucleotides or deoxyribonucleotides, or
analogs thereof. This term refers to the primary structure of the
molecule, and thus includes double- and single-stranded DNA, as
well as double- and single-stranded RNA. It also includes modified
polynucleotides such as methylated and/or capped
polynucleotides.
[0071] "Recombinant," as applied to a polynucleotide, means that
the polynucleotide is the product of various combinations of
cloning, restriction and/or ligation steps, and other procedures
that result in a construct that is distinct from a polynucleotide
found in nature.
[0072] A "gene" refers to a polynucleotide or portion of a
polynucleotide comprising a sequence that encodes a protein. For
most situations, it is desirable for the gene to also comprise a
promoter operably linked to the coding sequence in order to
effectively promote transcription. Enhancers, repressors and other
regulatory sequences may also be included in order to modulate
activity of the gene, as is well known in the art. (See, e.g., the
references cited below).
[0073] The terms "polypeptide," "peptide," and "protein" are used
interchangeably to refer to polymers of amino acids of any length,
or derivatives. These terms also include proteins that are
post-translationally modified through reactions that include
glycosylation, acetylation and phosphorylation.
[0074] Peptide "fragment" means any fragment or portion of the
peptide.
[0075] The terms "variant" and "amino acid sequence variant" are
used interchangeably and designate polypeptides in which one or
more amino acids are added and/or substituted and/or deleted and/or
inserted at the N- or C-terminus or anywhere within the
corresponding native sequences used herein, the term "variant" is
interpreted to mean a polynucleotide or polypeptide that differs
from a reference polynucleotide or polypeptide respectively, but
retains essential properties. A typical variant of a polynucleotide
differs in nucleotide sequence from another, reference
polynucleotide. Changes in the nucleotide sequence of the variant
may or may not alter the amino acid sequence of a polypeptide
encoded by the reference polynucleotide. Nucleotide changes may
result in amino acid substitutions, additions, deletions, fusions
and truncations in the polypeptide encoded by the reference
sequence. A typical variant of a polypeptide differs in amino acid
sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more substitutions,
additions, deletions in any combination. A substituted or inserted
amino acid residue may or may not be one encoded by the genetic
code. A variant of a polynucleotide or polypeptide may be a
naturally occurring such as an allelic variant, or it may be a
variant that is not known to occur naturally. Non-naturally
occurring variants of polynucleotides and polypeptides may be made
by mutagenesis techniques, by direct synthesis, and by other
recombinant methods known to skilled artisans.
[0076] The terms "derivatizing" and "derivative" or "derivatized"
include processes and all resulting peptides or modified peptides,
respectively. Including those in which (1) the peptide or modified
peptide has a cyclic portion; for example, cross-linking between
cysteinyl residues within the modified peptide; (2) the peptide or
modified peptide is cross-linked or has a cross-linking site; for
example, the peptide or modified peptide has a cysteinyl residue
and thus forms cross-linked dimers in culture or in vivo; (3) one
or more peptidyl linkage is replaced by a non-peptidyl linkage; (4)
the N-terminus is replaced by --NRR.sup.1, NRC(O)R.sup.1,
--NRC(O)OR.sup.1, --NRS(O).sub.2 R.sup.1, --NHC(O)NHR, a
succinimide group, or substituted or unsubstituted
benzyloxycarbonyl-NH--, wherein R and R.sup.1 and the ring
substituents are as defined hereinafter; (5) the C-terminus is
replaced by --C(O)R.sup.2 or --NR.sup.3 R.sup.4 wherein R.sup.2,
R.sup.3 and R.sup.4 are as defined hereinafter; and (6) peptides or
modified peptides in which individual amino acid moieties are
modified through treatment with agents capable of reacting with
selected side chains or terminal residues.
[0077] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. The antibodies,
peptides or vectors used as vaccines of the present invention can
be administered to a patient at therapeutically effective doses to
treat (including prevention) heart disease and/or other muscular
contractile disorders. A therapeutically effective dose refers to
that amount of the compound sufficient to result in desired
treatment. As used herein, the term "antibody or antibodies"
includes polyclonal and monoclonal antibodies of any isotype (IgA,
IgG, IgE, IgD, IgM), or an antigen-binding portion thereof,
including but not limited to F(ab) and Fv fragments, single chain
antibodies, chimeric antibodies, humanized antibodies, and a Fab
expression library. "Antibody" refers to a polypeptide ligand
substantially encoded by an immunoglobulin gene or immunoglobulin
genes, or fragments thereof, which specifically binds and
recognizes an epitope (e.g., an antigen). The recognized
immunoglobulin--genes include the kappa and lambda light chain
constant region genes, the alpha, gamma, delta, epsilon and mu
heavy chain constant region genes, and the myriad immunoglobulin
variable region genes. Antibodies exist, e.g., as intact
immunoglobulins or as a number of well characterized fragments
produced by digestion with various peptidases. This includes, e.g.,
Fab' and F(ab)'.sub.2 fragments. The term "antibody," as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies or those synthesized de novo using
recombinant DNA methodologies. It also includes polyclonal
antibodies, monoclonal antibodies, chimeric antibodies and
humanized antibodies. "Fc" portion of an antibody refers to that
portion of an immunoglobulin heavy chain that comprises one or more
heavy chain constant region domains, CH, CH.sub.2 and CH.sub.3, but
does not include the heavy chain variable region.
[0078] As used herein, the term "fragment", as applied to an
antibody means any fragment of the antibody that includes the
antigenic determinant/epitope to which the complete antibody binds,
including Fab, Fab', F(ab).sub.2, and F(ab').sub.2 fragments.
[0079] As used herein, the term "substantially pure or purified"
describes a compound (e.g., a protein or polypeptide) which has
been separated from components which naturally accompany it.
Typically, a compound is substantially pure when at least 10%, more
preferably at least 20%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 75%, more
preferably at least 90%, and even more preferably at least 99%, of
the total material (by volume, by wet or dry weight, or by mole
percent or mole fraction) in a sample is the compound of interest.
Purity can be measured by any appropriate method. In the case of
polypeptides, for example, purity can be measured by column
chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis. A compound such as a protein is also substantially
purified when it is essentially free of naturally associated
components or when it is separated from the native contaminants
which accompany it in its natural state.
[0080] A "substantially pure nucleic acid or purified", as used
herein, refers to a nucleic acid sequence, segment, or fragment
which has been purified from the sequences which flank it in a
naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment such as the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA, which
has been purified from proteins which naturally accompany it in the
cell.
[0081] A "promoter," as used herein, refers to a polynucleotide
sequence that controls transcription of a gene or coding sequence
to which it is operably linked. A large number of promoters,
including constitutive, inducible and repressible promoters, from a
variety of different sources, are well known in the art and are
available as or within cloned polynucleotide sequences (from, e.g.,
depositories such as the ATCC as well as other commercial or
individual sources).
[0082] An "enhancer," as used herein, refers to a polynucleotide
sequence that enhances transcription of a gene or coding sequence
to which it is operably linked. A large number of enhancers, from a
variety of different sources are well known in the art and
available as or within cloned polynucleotide sequences (from, e.g.,
depositories such as the ATCC as well as other commercial or
individual sources). A number of polynucleotides comprising
promoter sequences (such as the commonly-used CMV promoter) also
comprise enhancer sequences. "Operably linked" refers to a
juxtaposition, wherein the components so described are in a
relationship permitting them to function in their intended manner.
A promoter is operably linked to a coding sequence if the promoter
controls transcription of the coding sequence. Although an operably
linked promoter is generally located upstream of the coding
sequence, it is not necessarily contiguous with it. An enhancer is
operably linked to a coding sequence if the enhancer increases
transcription of the coding sequence. Operably linked enhancers can
be located upstream, within or downstream of coding sequences. A
polyadenylation sequence is operably linked to a coding sequence if
it is located at the downstream end of the coding sequence such
that transcription proceeds through the coding sequence into the
polyadenylation sequence.
[0083] A "replicon" refers to a polynucleotide comprising an origin
of replication which allows for replication of the polynucleotide
in an appropriate host cell. Examples include replicons of a target
cell into which a heterologous nucleic acid might be integrated
(e.g., nuclear and mitochondrial chromosomes), as well as
extrachromosomal replicons (such as replicating plasmids and
episomes).
[0084] In accordance with the invention, the antibodies of the
invention are also used as diagnostic agents which detect muscle
contractile disorders, especially, for example, in the heart. In
one embodiment, any of the above-described molecules can be
labeled, either detectably, as with a radioisotope, a paramagnetic
atom, a fluorescent moiety, an enzyme, etc. in order to facilitate
its detection in, for example, in situ or in vivo assays. The
molecules may be labeled with reagents such as biotin, in order to,
for example, facilitate their recovery, and/or detection.
[0085] As used herein, "inotropic agents" or "inotropic antibodies"
will be used interchangeably and refers to the effect such agents
produce, i.e. improves cardiac output by increasing the force of
myocardial muscle contraction. "Positive inotropic effect" means
that the contractility of the cells is enhanced in a dose-dependent
manner. A positive inotropic effect-producing amount of antibodies
or peptides of the invention can be administered to a "mammalian
host" (e.g., a human) to treat cardiac malfunction (e.g.,
congestive heart failure, paroxysmal atrial tachycardia, atrial
fibrillation and flutter). Administration can be either enteral
(i.e., oral) or parenteral (e.g., via intravenous, subcutaneous or
intramuscular injection).
[0086] As used herein, the term "antibody" refers to a polypeptide
or group of polypeptides which are comprised of at least one
binding domain. An antibody binding domain is formed from the
folding of variable domains of an antibody molecule to form
three-dimensional binding spaces with an internal surface shape and
charge distribution complementary to the features of an antigenic
determinant of an antigen. This allows an immunological reaction
with the antigen. Antibodies include recombinant proteins
comprising the binding domains, as wells as fragments, including
Fab, Fab', F(ab).sub.2, and F(ab').sub.2 fragments.
[0087] The term "polyclonal" refers to antibodies that are
heterogeneous populations of antibody molecules derived from the
sera of animals immunized with an antigen or an antigenic
functional derivative thereof. For the production of polyclonal
antibodies, various host animals may be immunized by injection with
the antigen. Various adjuvants may be used to increase the
immunological response, depending on the host species.
[0088] "Monoclonal antibodies" are substantially homogenous
populations of antibodies to a particular antigen. They may be
obtained by any technique which provides for the production of
antibody molecules by continuous cell lines in culture. Monoclonal
antibodies may be obtained by methods known to those skilled in the
art. See, for example, Kohler, et al., Nature 256:495-497, 1975,
and U.S. Pat. No. 4,376,110.
[0089] As used herein, an "antigenic determinant" is the portion of
an antigen molecule that determines the specificity of the
antigen-antibody reaction. An "epitope" also refers to an antigenic
determinant of a polypeptide and is used interchangeably herein. An
epitope can comprise as few as 3 amino acids in a spatial
conformation which is unique to the epitope. Generally an epitope
consists of at least 6 such amino acids, and more usually at least
8-10 such amino acids. Methods for determining the amino acids
which make up an epitope include x-ray crystallography,
2-dimensional nuclear magnetic resonance, and epitope mapping e.g.
the Pepscan method described by H. Mario Geysen et al. 1984. Proc.
Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO
84/03564; and PCT Publication No. WO 84/03506.
[0090] The phrase "binds" to an antibody or "specific binding" or
"selective binding" when referring to a protein or peptide, refers
to a binding reaction that is determinative of the presence of the
protein in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies or other compounds bind to a particular
protein or peptide at least two times the background and do not
substantially bind in a significant amount to other proteins or
peptides present in the sample. Specific binding to an antibody
under such conditions may require an antibody that is selected for
its specificity for a particular protein. For example, polyclonal
antibodies raised to marker "X" from specific species such as rat,
mouse, or human can be selected to obtain only those polyclonal
antibodies that are specifically immunoreactive with marker "X" and
not with other proteins, except for polymorphic variants and
alleles of marker "X". This selection may be achieved by
subtracting out antibodies that cross-react with marker "X"
molecules from other species. A variety of immunoassay formats may
be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A
Laboratory Manual (1988), for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity). Typically a specific or selective reaction will
be at least twice background signal or noise and more typically
more than 10 to 100 times background.
[0091] "Immunoassay" is an assay that uses an antibody to
specifically bind an antigen (e.g., a marker). The immunoassay is
characterized by the use of specific binding properties of a
particular antibody to isolate, target, and/or quantify the
antigen.
[0092] In another preferred embodiment, where the antibodies or
their fragments are intended for therapeutic purposes, it is
desirable to "humanize" them in order to attenuate any immune
reaction. Humanized antibodies may be produced, for example by
replacing an immunogenic portion of an antibody with a
corresponding, but non-immunogenic portion (i.e. chimeric
antibodies) (Robinson, R. R. et al., International Patent
Publication PCT/U.S. 86/02269; Akira, K. et al., European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison, S. L. et al., European Patent Application
173,494; Neuberger, M. S. et al., PCT Application WO 86/01533;
Cabilly, S. et al., European Patent Application 125,023; Better, M.
et al., Science 240:1041-1043 (1988); Liu, A. Y. et al. Proc. Natl.
Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol.
139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA
84:214-218 (1987); Nishimura, Y. et al., Canc. Res. 47:999-1005
(1987); Wood, C. R. et al., Nature 314:446-449 (1985)); Shaw et
al., J. Natl. Cancer Inst. 80:1553-1559 (1988); all of which
references are incorporated herein by reference). General reviews
of "humanized" chimeric antibodies are provided by Morrison, S. L.
(Science, 229:1202-1207 (1985)) and by Oi, V. T. et al.,
BioTechniques 4:214 (1986); which references are incorporated
herein by reference).
[0093] Suitable "humanized" antibodies can alternatively be
produced by CDR or CEA substitution (Jones, P. T. et al., Nature
321:552-525 (1986); Verhoeyan et al., Science 239:1534 (1988);
Beidler, C. B. et al., J. Immunol. 141:4053-4060 (1988); all of
which references are incorporated herein by reference).
[0094] As used herein, the term "humanized" antibody refers to a
molecule that has its CDRs (complementarily determining regions)
derived from a non-human species immunoglobulin and the remainder
of the antibody molecule derived mainly from a human
immunoglobulin. The term "antibody" as used herein, unless
indicated otherwise, is used broadly to refer to both antibody
molecules and a variety of antibody derived molecules. Such
antibody derived molecules have at least one variable region
(either a heavy chain of light chain variable region) and include
molecules such as Fab fragments, Fab' fragments, F(ab').sub.2
fragments, Fd fragments, Fab' fragments, Fd fragments, Fabc
fragments, Sc antibodies (single chain antibodies), diabodies,
individual antibody light chains, individual antibody heavy chains,
chimeric fusions between antibody chains and other molecules, and
the like.
[0095] The term "variable region" as used herein in reference to
immunoglobulin molecules has the ordinary meaning given to the term
by the person of ordinary skill in the act of immunology. Both
antibody heavy chains and antibody light chains may be divided into
a "variable region" and a "constant region". The point of division
between a variable region and a heavy region may readily be
determined by the person of ordinary skill in the art by reference
to standard texts describing antibody structure, e.g., Kabat et al
"Sequences of Proteins of Immunological Interest: 5th Edition" U.S.
Department of Health and Human Services, U.S. Government Printing
Office (1991).
[0096] The present invention provides humanized antibody molecules
specific for antigenic determinants or epitopes defined by peptides
having an amino acid sequence DVEDSYGQQWTYEQR, RSATEEEPPNDD,
KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN, and fragments,
derivatives and variants thereof. However, the invention is not
limited to these sequences but applies to any sequence in which
antibodies can bind resulting in an increase in sodium potassium
ATPase activity, and in the case of SSA401, in cardiac positive
inotropy. In accordance with the present invention, the humanized
antibodies are comprised of antigen specific regions in which at
least parts of the CDRs of the heavy and/or light chain variable
regions of a human antibody (the receptor antibody) have been
substituted by analogous parts of CDRs of a murine monoclonal
antibody. In a preferred embodiment of the subject invention, the
CDR regions of the humanized antibodies are derived from rabbits as
described in the examples which follow. Some of the humanized
antibodies described herein contain some alterations of the
acceptor antibody, i.e., human, heavy and/or light chain variable
domain framework regions that are necessary for retaining binding
specificity of the donor monoclonal antibody. In other words, the
framework region of some embodiments the humanized antibodies
described herein does not necessarily consist of the precise amino
acid sequence of the framework region of a natural occurring human
antibody variable region, but contains various substitutions that
improve the binding properties of a humanized antibody region that
is specific for the same target as the NKA activity-increasing
antibodies. A minimal number of substitutions are made to the
framework region in order to avoid large-scale introductions of
non-human framework residues and to ensure minimal immunogenicity
of the humanized antibody in humans.
[0097] The humanized antibodies compositions of the invention or
other therapeutic agents of the invention may be administered to a
patient in a variety of ways. In some embodiments the
pharmaceutical compositions may be administered parenterally, i.e.,
subcutaneously, intramuscularly or intravenously. Thus, this
invention provides compositions for parenteral administration which
comprise a solution of the human monoclonal antibody or a cocktail
thereof dissolved in an acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used, e.g., water,
buffered water, 0.4% saline, 0.3% glycine and the like. These
solutions are sterile and generally free of particulate matter.
These compositions may be sterilized by conventional, well known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate, etc. The concentration of
antibody in these formulations can vary widely, e.g., from less
than about 0.5%, usually at or at least about 1% to as much as 15
or 20% by weight and will be selected primarily based on fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected. Local injection or infusions can also
be used.
[0098] Actual methods for preparing parenterally administrable
compositions and adjustments necessary for administration to
subjects will be known or apparent to those skilled in the art and
are described in more detail in, for example, Remington's
Pharmaceutical Science, 15th Ed., Mack Publishing Company, Easton,
Pa. (1980), which is incorporated herein by reference.
[0099] As used herein, the term "pharmaceutically acceptable
carrier" encompasses any of the standard pharmaceutical carriers,
such as a phosphate buffered saline solution, water, and emulsions,
such as an oil/water or water/oil emulsion, and various types of
wetting agents. The compositions also can include stabilizers and
preservatives. For examples of carriers, stabilizers and adjuvants,
see Martin Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co.,
Easton (1975)).
[0100] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED.50. Compounds exhibiting
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects can be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0101] Data obtained from cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage can vary within this range depending upon-the
dosage form employed and the route of administration utilized. For
any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound, which achieves
a half-maximal inhibition of symptoms) as determined in cell
culture. Such information can be used to accurately determine
useful doses in humans. Levels in plasma can be measured, for
example, by high performance liquid chromatography. A typical daily
dose for the therapeutic molecules of the invention (i.e.,
antibodies, peptides, vectors encoding peptides) of the present
invention might range from about 1 microgram/kg to about 100 mg/kg
of patient body weight or more per day, depending on the factors
mentioned above, preferably about 10 microgram/kg/day to 10
mg/kg/day.
[0102] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates can be formulated for administration by intra venous,
intranasal or oral, buccal, parenteral or rectal
administration.
[0103] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate. talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration can be
suitably formulated to give controlled release of the active
compound. For buccal administration the compositions can take the
form of tablets or lozenges formulated in conventional manner.
[0104] The compounds can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
or dispersing agents. Alternatively, the active ingredient can be
in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use. The compounds can also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides.
[0105] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0106] The compositions can, if desired, be presented in a pack or
dispenser device that can contain one or more unit dosage forms
containing the active ingredient. The pack can for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device can be accompanied by instructions for
administration.
[0107] The treatment of heart diseases and contractile disorders
can be monitored bin various ways, including echography and
electrocardiograms. Echocardiography is the preferred method of
monitoring treatment using the molecules of the invention.
[0108] The invention also provides for vectors which are used for
treating a patient suffering from or susceptible heart disease. As
used herein, a "vector" (sometimes referred to as gene delivery or
gene transfer "vehicle") refers to a macromolecule or complex of
molecules comprising a polynucleotide to be delivered to a host
cell, either in vitro or in vivo. The polynucleotide to be
delivered may comprise a coding sequence of interest in gene
therapy. Vectors include, for example, viral vectors (such as
adenoviruses ("Ad"), adeno-associated viruses (AAV), and
retroviruses), liposomes and other lipid-containing complexes, and
other macromolecular complexes capable of mediating delivery of a
polynucleotide to a host cell. Vectors can also comprise other
components or functionalities that further modulate gene delivery
and/or gene expression, or that otherwise provide beneficial
properties to the targeted cells. As described and illustrated in
more detail below, such other components include, for example,
components that influence binding or targeting to cells (including
components that mediate cell-type or tissue-specific binding);
components that influence uptake of the vector nucleic acid by the
cell; components that influence localization of the polynucleotide
within the cell after uptake (such as agents mediating nuclear
localization); and components that influence expression of the
polynucleotide. Such components also might include markers, such as
detectable and/or selectable markers that can be used to detect or
select for cells that have taken up and are expressing the nucleic
acid delivered by the vector. Such components can be provided as a
natural feature of the vector (such as the use of certain viral
vectors which have components or functionalities mediating binding
and uptake), or vectors can be modified to provide such
functionalities. A large variety of such vectors are known in the
art and are generally available (see, e.g., the various references
cited below).
[0109] As used herein, the term "administering a molecule to a
cell" (e.g., an expression vector, nucleic acid, peptide, a
delivery vehicle, agent, and the like) refers to transducing,
transfecting, microinjecting, electroporating, or shooting, the
cell with the molecule. In some aspects, molecules are introduced
into a target cell by contacting the target cell with a delivery
cell (e.g., by cell fusion or by lysing the delivery cell when it
is in proximity to the target cell). This term is to be
distinguished from administering a composition to a patient.
[0110] A cell has been "transformed", "transduced", or
"transfected" by exogenous or heterologous nucleic acids when such
nucleic acids have been introduced inside the cell. Transforming
DNA may or may not be integrated (covalently linked) with
chromosomal DNA making up the genome of the cell. In prokaryotes,
yeast, and mammalian cells for example, the transforming DNA may be
maintained on an episomal element, such as a plasmid. In a
eukaryotic cell, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it
is inherited by daughter cells through chromosome replication. This
stability is demonstrated by the ability of the eukaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the transforming DNA. A "clone" is a
population of cells derived from a single cell or common ancestor
by mitosis. A "cell line" is a clone of a primary cell that is
capable of stable growth in vitro for many generations (e.g., at
least about 10.
[0111] As used herein, "molecule" is used generically to encompass
any vector, antibody, protein, drug and the like which are used in
therapy and can be detected in a patient by the methods of the
invention. For example, multiple different types of nucleic acid
delivery vectors encoding different types of genes which may act
together to promote a therapeutic effect, or to increase the
efficacy or selectivity of gene transfer and/or gene expression in
a cell. The nucleic acid delivery vector may be provided as naked
nucleic acids or in a delivery vehicle associated with one or more
molecules for facilitating entry of a nucleic acid into a cell.
Suitable delivery vehicles include, but are not limited to:
liposomal formulations, polypeptides; polysaccharides;
lipopolysaccharides, viral formulations (e.g., including viruses,
viral particles, artificial viral envelopes and the like), cell
delivery vehicles, and the like.
[0112] A "recombinant viral vector" refers to a viral vector
comprising one or more heterologous genes or sequences. Since many
viral vectors exhibit size-constraints associated with packaging,
the heterologous genes or sequences are typically introduced by
replacing one or more portions of the viral genome. Such viruses
may become replication-defective, requiring the deleted function(s)
to be provided in trans during viral replication and encapsidation
(by using, e.g., a helper virus or a packaging cell line carrying
genes necessary for replication and/or encapsidation) (see, e.g.,
the references and illustrations below). Modified viral vectors in
which a polynucleotide to be delivered is carried on the outside of
the viral particle have also been described (see, e.g., Curiel, D
T, et al. PNAS 88: 8850-8854, 1991).
[0113] Viral "packaging" as used herein refers to a series of
intracellular events that results in the synthesis and assembly of
a viral vector. Packaging typically involves the replication of the
"pro-viral genome", or a recombinant pro-vector typically referred
to as a "vector plasmid" (which is a recombinant polynucleotide
than can be packaged in an manner analogous to a viral genome,
typically as a result of being flanked by appropriate viral
"packaging sequences"), followed by encapsidation or other coating
of the nucleic acid. Thus, when a suitable vector plasmid is
introduced into a packaging cell line under appropriate conditions,
it can be replicated and assembled into a viral particle. Viral
"rep" and "cap" genes, found in many viral genomes, are genes
encoding replication and encapsidation proteins, respectively. A
"replication-defective" or "replication-incompetent" viral vector
refers to a viral vector in which one or more functions necessary
for replication and/or packaging are missing or altered, rendering
the viral vector incapable of initiating viral replication
following uptake by a host cell. To produce stocks of such
replication-defective viral vectors, the virus or pro-viral nucleic
acid can be introduced into a "packaging cell line" that has been
modified to contain genes encoding the missing functions which can
be supplied in trans). For example, such packaging genes can be
stably integrated into a replicon of the packaging cell line or
they can be introduced by transfection with a "packaging plasmid"
or helper virus carrying genes encoding the missing functions.
[0114] A "detectable marker gene" is a gene that allows cells
carrying the gene to be specifically detected (e.g., distinguished
from cells which do not carry the marker gene). A large variety of
such marker genes are known in the art. Preferred examples thereof
include detectable marker genes which encode proteins appearing on
cellular surfaces, thereby facilitating simplified and rapid
detection and/or cellular sorting. By way of illustration, the lacZ
gene encoding beta-galactosidase can be used as a detectable
marker, allowing cells transduced with a vector carrying the lacZ
gene to be detected by staining, as described below.
[0115] A "selectable marker gene" is a gene that allows cells
carrying the gene to be specifically selected for or against, in
the presence of a corresponding selective agent. By way of
illustration, an antibiotic resistance gene can be used as a
positive selectable marker gene that allows a host cell to be
positively selected for in the presence of the corresponding
antibiotic. Selectable markers can be positive, negative or
bifunctional. Positive selectable markers allow selection for cells
carrying the marker, whereas negative selectable markers allow
cells carrying the marker to be selectively eliminated. A variety
of such marker genes have been described, including bifunctional
(i.e. positive/negative) markers (see, e.g., WO 92/08796, published
May 29, 1992, and WO 94/28143, published Dec. 8, 1994). Such marker
genes can provide an added measure of control that can be
advantageous in gene therapy contexts. "Treatment" or "therapy" as
used herein also refers to administering, to an individual patient,
agents that are capable of eliciting a prophylactic, curative or
other beneficial effect in the individual.
[0116] "Gene therapy" as used herein refers to administering, to an
individual patient, vectors comprising a therapeutic gene, such as
the vectors carrying one or more of the peptides DVEDSYGQQWTYEQR,
RSATEEEPPNDD, KRQPRNPKTDKLVNE, VPAISLAYEQAESD and HLLGIRETWDDRWIN,
or to fragments, derivatives or variants thereof.
[0117] A "therapeutic polynucleotide" or "therapeutic gene" refers
to a nucleotide sequence that is capable, when transferred to an
individual, of eliciting a prophylactic, curative or other
beneficial effect in the individual. Such as expressing one or more
of the peptide antigenic epitopes described herein which in turn
elicit an immune response from the host.
[0118] The practice of the present invention can suitably employ,
unless otherwise indicated, conventional techniques of molecular
biology and the like, which are within the skill of the art. Such
techniques are explained fully in the literature. See e.g.,
Molecular Cloning: A Laboratory Manual, (J. Sambrook et al., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current
Protocols in Molecular Biology (F. Ausubel et al. eds., 1987 and
updated); Essential Molecular Biology (T. Brown ed., IRL Press
1991); Gene Expression Technology (Goeddel ed., Academic Press
1991); Methods for Cloning and Analysis of Eukaryotic Genes (A.
Bothwell et al. eds., Bartlett Publ. 1990); Gene Transfer and
Expression (M. Kriegler, Stockton Press 1990); Recombinant DNA
Methodology (R. Wu et al. eds., Academic Press 1989); PCR: A
Practical Approach (M. McPherson et al., IRL Press at Oxford
University Press 1991); Cell Culture for Biochemists (R. Adams ed.,
Elsevier Science Publishers 1990); Gene Transfer Vectors for
Mammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian
Cell Biotechnology (M. Butler ed., 1991); Animal Cell Culture (J.
Pollard et al. eds., Humana Press 1990); Culture of Animal Cells,
2nd Ed. (R. Freshney et al. eds., Alan R. Liss 1987); Flow
Cytometry and Sorting (M. Melamed et al. eds., Wiley-Liss 1990);
the series Methods in Enzymology (Academic Press, Inc.); Techniques
in Immunocytochemistry, (G. Bullock & P. Petrusz eds., Academic
Press 1982, 1983, 1985, 1989); Handbook of Experimental Immunology,
(D. Weir & C. Blackwell, eds.); Cellular and Molecular
Immunology (A. Abbas et al., W.B. Saunders Co. 1991, 1994); Current
Protocols in Immunology (J. Coligan et al. eds. 1991); the series
Annual Review of Immunology; the series Advances in Immunology;
Oligonucleotide Synthesis (M. Gait ed., 1984); and Animal Cell
Culture (R. Freshney ed., IRL Press 1987).
[0119] Preferred vectors for use in the present invention include
viral vectors, lipid-based vectors and other vectors that are
capable of delivering DNA to non-dividing cells in vivo. Presently
preferred are viral vectors, particularly replication-defective
viral vectors (including, for example replication-defective
adenovirus vectors and adeno-associated virus (AAV) vectors. For
ease of production and use in the present invention,
replication-defective adenovirus vectors are presently most
preferred.
[0120] "Gene delivery," "gene transfer," and the like as used
herein, are terms referring to the introduction of an exogenous
polynucleotide (sometimes referred to as a "transgenes") into a
host cell, irrespective of the method used for the introduction.
Such methods include a variety of well-known techniques such as
vector-mediated gene transfer (by, e.g., viral
infection/transfection, or various other protein-based or
lipid-based gene delivery complexes) as well as techniques
facilitating the delivery of "naked" polynucleotides (such as
electroporation, "gene gun" delivery and various other techniques
used for the introduction of polynucleotides). The introduced
polynucleotide may be stably or transiently maintained in the host
cell. Stable maintenance typically requires that the introduced
polynucleotide either contains an origin of replication compatible
with the host cell or integrates into a replicon of the host cell
such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear
or mitochondrial chromosome. A number of vectors are known to be
capable of mediating transfer of genes to mammalian cells, as is
known in the art and described herein. Targeted vectors include
vectors (such as viruses, non-viral protein-based vectors and
lipid-based vectors) in which delivery results in transgene
expression that is relatively limited to particular host cells or
host cell types. By way of illustration, therapeutic molecules, for
example, nucleic acid sequences encoding for the peptides of the
invention, to be delivered to a patient can be operably linked to
heterologous tissue-specific promoters thereby restricting
expression to cells in that particular tissue.
[0121] "In vivo" gene delivery, gene transfer, gene therapy and the
like as used herein, are terms referring to the introduction of a
vector comprising an exogenous polynucleotide directly into the
body of an organism, such as a human or non-human mammal, whereby
the exogenous polynucleotide is introduced to a cell of such
organism in vivo.
[0122] When vectors are used to express peptide epitopes for
example to stimulate production of antibody for use in treating or
preventing diseases associated with low NKA activity, including
heart disease or contractile disorders, they are sometimes
administered systemically. Other times they are administered
locally for example by injection into a blood vessel directly
supplying the target tissue. For example, if it is the heart, then
they are injected into a vessel supplying the myocardium,
preferably by injection into a coronary artery. Such injection is
preferably achieved by catheter introduced substantially (typically
at least about 1 cm) within the ostium of one or both coronary
arteries or one or more saphenous veins or internal mammary artery
grafts or other conduits delivering blood to the myocardium. By
injecting the vector stock, preferably containing no wild-type
virus, deeply into the lumen of an artery (or grafts and other
vascular conduits), and preferably in an amount of about
10.sup.7-13 viral particles as determined by optical densitometry
(more preferably 10.sup.9-11 viral particles), it is possible to
locally transfect a desired number of cells with genes that encode
proteins that regulate cell NKA activity, such as, for example, the
peptides discussed herein. This maximizes the therapeutic efficacy
of gene transfer, and minimizes undesirable effects at other sites
such as the possibility of an inflammatory response to viral
proteins. For example, vector constructs that are specifically
targeted to the myocardium, such as vectors incorporating
myocardial-specific binding or uptake components, and/or which
incorporate inotropic molecules, for example, the peptides
described above, that are under the control of myocardial-specific
transcriptional regulatory sequences (e.g., ventricular
myocyte-specific promoters) can be used in place of or, preferably,
in addition to such directed injection techniques as a means of
further restricting expression to the myocardium, especially the
ventricular myocytes. For vectors that can elicit an immune
response, it is preferable to inject the vector directly into a
blood vessel supplying the targeted cells or tissue, although the
additional techniques for restricting the potential for non-target
expression can also be employed.
[0123] The invention also provides for methods for identifying
peptides and antibodies that activate NKA and therefore come within
the scope of the invention. To prepare an antibody that
specifically binds to a region of the NKA, purified peptides or
their nucleic acid sequences representing the different subunits of
NKA can be used. Using these purified peptides or their nucleic
acid sequences, antibodies that specifically bind to a desired
peptide can be prepared using any suitable method known in the art.
See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow
& Lane, Antibodies: A Laboratory Manual (1988); Goding,
Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and
Kohler & Milstein, Nature 256:495-497 (1975). Such techniques
include, but are not limited to, antibody preparation by selection
of antibodies from libraries of recombinant antibodies in phage or
similar vectors, as well as preparation of polyclonal and
monoclonal antibodies by immunizing animals (see, e.g., Huse et al,
Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546
(1989)); humanized antibodies; production of antibodies by any of
the methods discussed above. After the antibody is provided, the
specificity of the antibody can be detected using any of suitable
immunological binding assays known in the art (see, e.g., U.S. Pat.
Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). Useful assays
include, for example, an enzyme immune assay (EIA) such as
enzyme-linked immunosorbent assay (ELISA), a radioimmune assay
(RIA), a Western blot assay, or a slot blot assay. These methods
are also described in, e.g., Methods in Cell Biology: Antibodies in
Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical
Immunology (Stites & Terr, eds., 7th ed. 1991); and Harlow
& Lane, supra.
[0124] To determine whether these identified antibodies increase
NKA activity or are positive inotropic agents, standard assays such
as those described in the Examples which follow can be used. For
example, measurement of cell contraction assays; confocal Ca ion
imaging; NKA activity assays and the like.
EXAMPLES
Example 1
Preparation of Antibodies
[0125] Polyclonal antibodies SSA78, 95, 97, 401 and 412 were
generated against NKA in New Zealand white rabbits using KLH
(Keyhole Limpet Hemocyanin) as a peptide carrier. Sprague Dawley
rats were obtained from Charles River Laboratories. Protocols were
approved by the Animal Care and Use Committees of the University Of
Maryland School Of Medicine. The peptides were synthesized
according to the protein sequence reported (Schneider, J. W.,
Mercer, R. W., Caplan, M., Emanuel, J. R., Sweadner, K. J., Benz,
E. J., Levenson, R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82,
6357-6361; Xie, Z., Li, H., Liu, G., Wang, Y., Askari, A., Mercer,
R. W. (1994) Cloning of the dog Na/K-ATPase alpha 1-subunit. The Na
Pump. (Bamberg, S., and Schoner, W., Eds), pp. 49-52,
Springer-Verlag, New York, N.Y.; Shull, M. M., Lingrel, J. B.
(1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4039-4043). The
immunoglobulins (IgG) were purified through an affinity column
directed against the same synthetic peptide of the NKA. Purified
antibodies against peptide epitopes in NKATPase recognize both
denatured (by Western blots) and native NKATPase (by
immunocytostaining). Synthetic peptide was also utilized as the
specific peptide blockers for the antibodies.
Example 2
Isolation of Cardiac Myocytes
[0126] Ventricular cardiac myocytes were isolated from adult
Sprague-Dawley rats (2-3 months old, weight 225-300 g) using
standard enzymatic technique as described previously..sup.1
Briefly, following anaesthesia (sodium pentobarbital, 100 mg/kg),
the heart was quickly removed from the chest and aortic perfused at
constant pressure at 37.degree. C. for 3 min with a Ca.sup.2+-free
bicarbonate-based buffer containing 120 mM NaCL, 5.4 mM MgSO.sub.4,
1.2 mM NaH.sub.2PO.sub.4, 5.6 mM glucose, 20 mM NaHCO.sub.3, and 5
mM taurine, in the presence of O.sub.2 (95%)/CO.sub.2 (5%).
Enzymatic digestion was initiated by adding collagenase
(Worthington Type II, 1 mg/ml) to the perfusion solution. Calcium
(50 .mu.M) was added to the enzyme solution when the heart became
swollen. About 7 min later, the left ventricle was quickly removed,
cut into several pieces, and further digested on a shaker (60-70
rpm) for 10 min in the same enzyme solution. The supernatant
containing the dispersed myocytes was filtered into a test tube and
gently centrifuged at 500 rpm for 1 min. The cell pellet was then
promptly resuspended in a solution containing 0.125 mM Ca.sup.2+.
The supernatant was aspirated after the myocytes were pelleted by
gravity for 10 min, and the myocytes were resuspended in a solution
containing 0.25 mM Ca.sup.2+. The shake-harvest procedure was
repeated several times until all the pieces were digested. For
freshly isolated cells, myocytes were suspended in HEPES-buffer
consisting of 1 mM CaCL2, 0.137 mM NaCL, 5.4 mM KCL, 15 mM
dextrose, 1.3 mM MgSO4, 1.2 mM NaH2PO4, and 20 mM HEPES, pH
7.4.
Example 3
Measurement of Cell Contraction (Cell Shortening)
[0127] Myocytes were placed on an inverted microscope (Zeiss model
IM-35), superfused with HEPES-buffered solution at a flow rate of
1.8 ml/min, and electrically stimulated at 0.5 Hz at 23.degree. C.
Cell length was monitored from the bright-field image (650 nm to
750 nm red light illumination) by an optical edge-tracking method
using a photodiode array (model 1024 SAQ, Reticon) with a 3-ms time
resolution. Contraction amplitude was measured as the percentage of
shortening of cell length.
Example 4
Immunoprecipitation, Blotting, and Immunofluorescent Staining
[0128] Isolated rat heart cells were incubated in 1 ml cold lysis
buffer containing 150 mM NaCl, 10 mM EDTA, 1% NP-40, 1 mM
Na.sub.3VO.sub.4, and 1.times. protease inhibitor cocktail as
described [12], with or without antibody. Immunoprecipitates were
boiled for 5 min and loaded on a 7% SDS gel, and transferred from
the SDS gel to a nitrocellulose membrane. The nitrocellulose
membrane was blocked with 5% nonfat milk for 1 hour and incubated
with SSA412 or other control antibodies overnight at 4.degree. C.
Alkaline phosphatase conjugated secondary antibody (1:2500) was
then incubated with the membrane for 1 hour and washed 3 times with
TBS in 0.05% Tween-20. Color was developed using a reagent
containing a mixture of NBT and BCIP (Promega Corporation, Wis.).
For immunofluorescent staining, rat myocytes were frozen and cut on
a cryostat. Sections (8 .mu.m) of each tissue were blocked with 1%
bovine serum albumin (BSA) and incubated with SSA412 (1:100) for 60
min in the presence or absence of 5, 10, and 20 mM ouabain. Washed
slides were evaluated after incubation with a FITC conjugated goat
anti-rabbit antibody (1:75).
Example 5
Intracellular Ca.sup.2+ Transients and Cell Contraction
[0129] Cardiac myocytes were isolated from adult Sprague-Dawley
rats, using standard enzymatic methods [13], and suspended in
buffer containing (in mM) 137 NaCl, 5.4 KCl, 15 dextrose, 1.3
MgSO.sub.4, 1.2 NaH.sub.2PO.sub.4, 1 CaCl.sub.2, and 20 HEPES, pH
7.4. Myocytes were loaded with 50 .mu.g of Indo-1/AM for 10 min,
washed and resuspended in HEPES-buffered solution in the presence
of 1 mM Ca.sup.2+, then stored in the dark at room temperature (RT)
for 60 min before use [14]. Cells were placed on the stage of a
modified inverted microscope equipped for simultaneous recording of
both Indo-1 fluorescence and cell length. Cells were studied at
room temperature at a stimulation rate of 0.5 Hz, excited at
350-nm, and the ratio of 410:490 emission was determined to
quantify intracellular calcium. Cells were electrically stimulated
at 0.5 Hz at 25.degree. C. and monitored by optical edge-tracking
using photodiode array. Contraction amplitude was indexed by the
percent shortening of cell length.
Example 6
Isolation of Sarcolemmal Vesicles and Purification of NKA
[0130] Cardiac sarcolemmal (SL) vesicles were isolated from rat
hearts as reported previously [15]. The SL vesicles were tested
with saponin and found to be predominately right-side-out in
orientation. NKA was further purified as described [16]. Briefly,
rat SL vesicles (4.4 mg/ml) were titrated with 0.58 mg/ml of SDS in
the presence of 2 mM ATP at 20.degree. C. for 30 min and then
loaded on the top of a sucrose (W/W) step gradient (15%, 28.8%
& 37.3%) in a T.+-.60 tube and centrifuged at 40,000 rpm for 90
minutes. The fractions that contain NKA were collected and stored
at -70.degree. C.
Example 7
Enzyme Catalytic Activity
[0131] NKA activity was determined based on Jack Kyte's method as
previously described [17] under various experimental conditions as
indicated in FIG. 2. All NKA activities in different experiments
are ouabain-sensitive activity. Purified ouabain-resistant rat NKA
and ouabain-sensitive dog NKA were used to verify the data. Enzymes
were incubated with or without SSA412 for 60 min at room
temperature. The reaction was initiated by adding different
concentrations of MgATP in a final volume of 0.25 ml at 37.degree.
C. for 30 min and terminated by adding 0.75 ml quench solution and
0.025 ml developer. Color was developed for 30 minutes at room
temperature and the concentration of phosphate was then determined
at 700 nm using a spectrophotometer. NKA turnover number
(k.sub.cat) was calculated using the equation
k.sub.cat=V.sub.max/E.sub.t, where V.sub.max is the maximal
velocity (rate of reaction) and E.sub.t represents enzyme
concentration.
Example 8
Enzyme Phosphorylation
[0132] Purified rat or dog NKA was phosphorylated in 20 mM Tris/Cl
buffer (pH 7.4) in the presence of 100 mM Na.sup.+ without K.sup.+
or 20 mM K.sup.+ without Na.sup.+, 10 .mu.M MgATP, and 1 nM
[.gamma.-.sup.32P]ATP (3000 Ci/mmol) with or without SSA412 or
total rabbit IgG. The reaction was stopped at the end of the
indicated time interval by a quench solution at pH 2.0. Samples
were transferred to a 2.0 ml polypropylene tube with 0.45 .mu.m
cellulose acetate filter and washed three times. The radioactivity
of each sample was determined using a .beta.-scintillation counter.
The number of net .sup.32P bound to the enzyme was calculated and
compared with control samples in the presence of SSA412.
Example 9
Measuring L-type Ca.sup.2+ Currents and Myoplasmic Ca.sup.2+
Transients in Rat Neonatal Ventricular Myocytes
[0133] The effects of SSA412 antibody on L-type Ca.sup.2+ currents
and myoplasmic Ca.sup.2+ transients were measured in rat neonatal
ventricular myocytes grown on cover slips and voltage-clamped
(`whole-cell` patch-clamp). Myoplasmic Ca.sup.2+ was determined
from simultaneous confocal optical recording of fluo-4
fluorescence. FIG. 9 The left-hand column represents a control
myocyte, while the right-hand column shows results from a myocyte
previously exposed to SSA412 antibody (1.0 uM). FIG. 9A The voltage
pulse protocol, V.sub.m. A ramp preceded each test pulse (-10 mV,
100 ms) in order to suppress Na.sup.+ and T-type Ca.sup.2+
currents. FIG. 9B LTCC currents (I.sub.Ca), corrected for linear
capacitance and leak using a P/4 protocol. FIG. 9C Myoplasmic
Ca.sup.2+ during and after the test pulse. Panel C shows the
myoplasmic Ca.sup.2+ as determined from confocal line-scan (x vs.
t) images. FIG. 9D Averaged results of peak I.sub.Ca and peak
myoplasmic Ca.sup.2+ elevation in each group. Values represent the
mean (.+-.SEM).
Example 10
Cyclic AMP Assays
[0134] Isolated rat cardiac myocytes were treated without or with
the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX,
1 mM) as shown in FIG. 11 side by side for 30 min at room
temperature prior to being incubated with either active SSA412
antibody or boiled SSA412 (4 .mu.M each) for 30 min, or with b-AR
agonist, isoprotenol (1 mM) for 5 min as a positive control.
Formation of cAMP was assayed using .sup.3H-cAMP assay kit obtained
from Amersham (Arlington Heights, Ill.). Protein concentration was
measured using the Bradford method (Bio-Rad, Richmond, Calif.) with
bovine serum albumin as standard. a a': control samples; b and b':
with 1 micromolar isoprotenol; c and c': with denatured SSA412; d
and d': with active SSA412; a', b', c' and d': in the presence of 1
mM IBMX.
[0135] In the foregoing specification, the invention has been
described with reference to specific embodiments thereof. It will,
however, be evident that various modifications and changes may be
made thereto without departing from the broader spirit and scope of
the invention. The specification and drawings are, accordingly, to
be regarded in an illustrative rather than a restrictive sense.
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Sequence CWU 1
1
5115PRTArtificial SequenceActivation site of the (Na+K)-ATPase 1Lys
Arg Gln Pro Arg Asn Pro Lys Thr Asp Lys Leu Val Asn Glu1 5 10
15213PRTArtificial SequenceActivation site of the (Na+K)-ATPase
2Val Pro Ala Ser Leu Ala Tyr Glu Gln Ala Glu Ser Asp1 5
10312PRTArtificial SequenceActivation site of the (Na+K)-ATPase
3Arg Ser Ala Thr Glu Glu Glu Pro Pro Asn Asp Asp1 5
10415PRTArtificial SequenceActivation site of the (Na+K)-ATPase
4His Leu Leu Gly Ile Arg Glu Thr Trp Asp Asp Arg Trp Ile Asn1 5 10
15515PRTArtificial SequenceActivation site of the (Na+K)-ATPase
5Asp Val Glu Asp Ser Tyr Gly Gln Gln Trp Thr Tyr Glu Gln Arg1 5 10
15
* * * * *